Relation between ESR-detectable Cu(II) and superoxide dismutase activity

The relation between ESR-detectable Cu(II) and Cu,Zn-superoxide dismutase activity was examined. The Cu(II) spin numbers per one unit of SOD were 6.26 X 10(12) (+/- 0.51 X 10(12] spins in several preparations of recombinant human Cu,Zn-SOD, native placental, and erythrocyte SOD. Measurement could be performed over a wide range of pH (4.0-10.0), preferably at temperatures below -40 degrees C. The data obtained by this method correlated well to the results obtained by the method of Fridovich et al. using the xanthine-xanthine oxidase system (correlation coefficient 0.995). The specific activity of SOD was proportional to the Cu(II) content measured by ESR, but not to the total Cu content measured by atomic absorption. This indicates that it is important to measure the Cu(II) content for determining Cu,Zn-SOD activity.     

Synthesis, structure and biomimetic properties of Cu(II)-Cu(II) and Cu(II)-Zn(II) binuclear complexes: possible models for the chemistry of Cu-Zn superoxide dismutase

Four imidazolate-bridged binuclear copper(II)-copper(II) and copper(II)-zinc(II) complexes viz., [(Bipy)(2)Cu-Im-Cu(Bipy)(2)](ClO(4))(3).CH(3)OH, [(Phen)(2)Cu-Im-Cu(Phen)(2)](BF(4))(3).2CH(3)OH, [(Bipy)(2)Cu-Im-Zn(Bipy)(2)](BF(4))(3), and [(Phen)(2)Cu-Im-Zn(Phen)(2)](BF(4))(3), (Bipy=2,2'-Bipyridyl, Phen=1-10-Phenanthroline and Im=imidazolate ion) were synthesized as a possible models for superoxide dismutase (SOD). Complex [(Bipy)(2)Cu-Im-Cu(Bipy)(2)](ClO(4))(3).CH(3)OH has been structurally characterized. This complex crystallizes in the triclinic space group P1, with the unit parameters a=8.88(5) A, b=13.79(17) A, c=20.18(18) A, alpha=76.424(8)(o), beta=85.888(6)(o), gamma=82.213(7). The metal-nitrogen bond length from 1.972-2.273 A and the distance Cu-Cu is 5.92 A. The five-coordinate geometry about the copper(II) ion is square pyramidal. Magnetic moment and electron paramagnetic resonance (e.p.r.) spectral measurements of the homobinuclear complexes have shown an antiferromagnetic exchange interaction. From the e.p.r. and UV-Vis spectral measurement studies, these complexes have been found to be stable (pH 8.5-10.5 for 1, 10.5 for 2,3 and 8.5 for 4). These complexes catalyse the dismutation of superoxide radical (O(2)(-)) at biological pH. All the observations indicate that these complexes act as good possible models for superoxide dismutase.     

Inhibition of bovine plasma amine oxidase by superoxide dismutase active Cu(II) complexes

Aqueous Cu²⁺ and Cu(II) complexes of salicylate, lysine, and tyrosine decrease the rate of benzylamine oxidation by bovine plasma amine oxidase. Bissalicylato Cu(II) and Cu²⁺ inhibit non-competitively with respect to benzylamine. Lysine, tyrosine, Cu(EDTA)^2?, Zn²⁺, and Co²⁺ do not inhibit, and erythrocyte Cu, Zn superoxide dismutase shows only slight inhibition of the amine oxidase. The data are most consistent with an inhibitory mechanism involving dismutation of O₂^? by the Cu(II) complexes within a site relatively inaccessible to the enzyme superoxide dismutase. Excess lysine significantly decreases inhibition by the bis-lysine complex of Cu(II).     

Photoelectron transfer processes with ruthenium(II) polypyridyl complexes and Cu/Zn superoxide dismutase

The processes that are photoinduced by [Ru(bpz)(3)](2+) (bpz = 2,2'-bipyrazyl) in the presence of Cu/Zn superoxide dismutase (Cu/Zn SOD) are investigated by laser flash photolysis and electron paramagnetic resonance (EPR) spectroscopy; they are compared to those of the system [Ru(bpy)(3)(2+)-Cu/Zn SOD]. Although the mechanism is complicated, primary and secondary reactions can be evidenced. First, the excited [Ru(bpz)(3)](2+) complex is quenched reductively by Cu/Zn SOD with the production of a reduced complex and an oxidized enzyme. The oxidation site of Cu/Zn SOD is proposed to correspond to amino acids located on the surface of the protein. Afterward and only when this reductive electron transfer to the excited complex has produced enough oxidized protein, another electron-transfer process can be evidenced. In this case, however, the charge-transfer process takes place in the other direction, i.e., from the excited complex to the Cu(II) center of the SOD with the formation of Ru(III) and Cu(I) species. This proposed mechanism is supported by the fact that [Ru(bpy)(3)](2+), which is less photo-oxidizing than [Ru(bpz)(3)](2+), exhibits no photoreaction with Cu/Zn SOD. Because Ru(III) species are generated as intermediates with [Ru(bpz)(3)](2+), they are proposed to be responsible for the enhancement of [poly(dG-dC)](2) and [poly(dA-dT)](2) oxidation observed when Cu/Zn SOD is added to the [Ru(bpz)(3)](2+)-DNA system.     

E.s.r., visible and SOD studies of imidazolate bridged Cu(2)(II,II), Cu(II)Zn(II) and Cu(II)Ni(II) complexes with pentamethyldiethylenetriamine as capping ligand: a plausible model for superoxide dismutase

X-band e.s.r. and electronic spectra of imidazolate bridged homobinuclear Cu-Cu complex, [(PMDT)Cu-Im-Cu(PMDT)](ClO(4))(3) and heterobinuclear Cu-Zn and Cu-Ni complexes, viz. [(PMDT)Cu-Im-Zn(PMDT)](ClO(4))(3), [(PMDT)Cu-Im-Ni(PMDT)] (ClO(4))(3), where PMDT=pentamethyldiethylenetriamine, Im=Imidazolate ion and related mononuclear complexes, [(PMDT)Cu(OH(2))](2+) and [(PMDT)Cu(ImH)](2+) have been described. Superoxide dismutase activities of these complexes have also been measured.     

Reactivity of antiinflammatory and superoxide dismutase active Cu(II)-salicylates.

The activity of chelated Cu(II) with four different aspirin-like drugs in various superoxide dismutase assays was examined. Prior to these studies the oxidation state of the involved copper was measured by x-ray photoelectron spectrometry and was found to be +II throughout. All copper complexes were able to suppress the xanthine-xanthine oxidase mediated reduction of both cytochrome c and nitroblue tetrazolium as well as the formazan formation by KO2 in a specific manner. The hydroxylation of benzo-[alpha]-pyrene as well as the demethylation of 7-ethoxycoumarin using induced hepatic rat microsomes could be successfully inhibited by the employed Cu(II) chelates. Cu(II)-acetylsalicylate was the most active copper complex. Our findings support the proposal that Cu(II) chelates are the active forms of aspirin-like antiinflammatory agents.     

Copper(II) complexes as superoxide dismutase mimics: Synthesis, characterization, crystal structure and bioactivity of copper(II) complexes

Two new copper(II) complexes of the type [Cu(L)X₂), where L = (E)-N-phenyl-2-[phenyl (pyridine-2-yl)methylene]hydrazinecarboxamide X = Cl/Br have been synthesized and characterized by elemental analyses, FAB (fast atomic bombardment) magnetic measurements, electronic absorption, conductivity measurements cyclic voltammetry (CV) and Electron paramagnetic resonance (epr) spectroscopy. The structures of these complexes determined by single crystal X-ray crystallography show a distorted square based pyramidal (DSBP) geometry around copper(II) metal center. The distorted CuN₂OX (X = Cl/Br) basal plane in them is comprised of two nitrogen and one oxygen atoms of the meridionally coordinated ligand and a chloride or bromide ion and axial position is occupied by other halide ion. The epr spectra of these complexes in frozen solutions of DMSO showed a signal at g ca. 2. The trend in g-value (g_|| > g_⊥ > 2.00) suggest that the unpaired electron on copper(II) has d_x²-y² character. Biological activities in terms of superoxide dismutase (SOD) and antimicrobial properties of copper(II) complexes have also been measured. The superoxide dismutase activity reveals that these two complexes catalyze the fast disproportionation of superoxide in DMSO solution.     

Binuclear copper(II) complexes of tolfenamic: synthesis, crystal structure, spectroscopy and superoxide dismutase activity

The synthesis and characterization of copper(II) complexes with a potent non-steroidal anti-inflammatory drug, tolfenamic acid, Htolf, with formula [Cu(tolf)(2)L](2) (where L is H(2)O or DMF, N,N-dimethylformamide) were investigated. The crystal and molecular structure of [Cu(tolf)(2)(DMF)](2) was reported. Crystallographic data are as follows: monoclinic system, space group P2(1)/n with cell constants a=9.068(2) A, b=14.514(3) A, c=22.826(4) A, V=2948.9(10) A(3) and Z=2. The crystal structure consists of binuclear, quadruply bridged neutral molecule with a Cu-Cu bond length of 2.6075(19) A. The complex is self-assembled via C-H-pi intermolecular stacking interactions. Spectroscopic and electrochemical studies were reported. The superoxide dismutase activity is measured and compared with those of superoxide dismutase enzyme, SOD, the free ligand and related copper complexes with non-steroidal anti-inflammatory drugs, NSAIDs. IC(50) value was measured by the Fridovich test (1.97+/-0.17 microM), which showed that [Cu(tolf)(2)L](2) is a good superoxide scavenger.     

Serum superoxide dismutase 3 (extracellular superoxide dismutase) activity is a sensitive indicator of Cu status in rats

Sensitivity of the assay for Cu,Zn superoxide dismutase 3 (SOD3), the predominant form of SOD in serum, can be increased, and interferences caused by low-molecular-weight substances in the serum can be reduced by conducting the assay at pH 10 with xanthine/xanthine oxidase and acetylated cytochrome c (cyt c) as superoxide generator and detector, respectively. Serum SOD3 activity was assayed under these conditions in an experiment where weanling, male rats were fed diets for 6 weeks containing 3, 5 and 15 mg Zn/kg with dietary Cu set at 0.3, 1.5 and 5 mg Cu/kg at each level of dietary Zn. Serum SOD3 responded to changes in dietary Cu but not to changes in dietary Zn. A second experiment compared serum SOD3 activity to traditional indices of Cu status in weanling, male and female rats after they were fed diets containing, nominally, 0, 1, 1.5, 2, 2.5, 3 and 6 mg Cu/kg for 6 weeks. Serum SOD3 activity was significantly lower (P < .05) in male rats fed diets containing 0 and 1 mg Cu/kg and female rats fed diet containing 0 mg Cu/kg compared with rats fed diet containing 6 mg Cu/kg. These changes were similar to changes in liver Cu concentrations, liver cyt c oxidase (CCO) activity and plasma ceruloplasmin in males and females. Serum SOD3 activity was also strongly, positively correlated with liver Cu concentrations over the entire range of dietary Cu concentrations (R(2) = .942 in males, R(2) = .884 in females, P < .0001). Plots of serum SOD3 activity, liver Cu concentration, liver CCO activity and ceruloplasmin as functions of kidney Cu concentration all had two linear segments that intersected at similar kidney Cu concentrations (18-22 microg/g dry kidney in males, 15-17 microg/g dry kidney in females). These findings indicate that serum SOD3 activity is a sensitive index of Cu status.     

Superoxide dismutase activity of Cu(Tyr)2 and Cu, Co-erythrocuprein.

Crystalline Cu(Tyr)2 and homogeneous Cu2Co2-erythrocuprein were prepared. The reactivity of each chelated Cu2 compound with superoxide was studied by pulse radiolysis at pH 7.6 +/- 0.1 and compared with the reactivity of native erythrocuprein (superoxide dismutase). Superoxide anions were generated by a 40-ns pulse of 1.81-MeV electrons. The yield of O2 ranged between 6 - 60 muM. The kinetics of the spontaneous O2 decay were second order; in the presence of Cu2 complexes the reaction was first order with respect to O2. Taking into account the effect of the different Cu2 concentrations on the O2 decay, second-order rate constants for the reaction of chelated Cu2 with O2 were obtained. For an equivalent of Cu2 in either erythrocuprein or Cu, Co-erythrocuprein, a numerical value of 1.3 +/- 0.1 x 10(9) M-1S-1 was calculated. Surprisingly, the same value was obtained employing Cu(Tyr)2. The highest rate constant was measured for the hydrated Cu2 (2.7 x 10(9) M-1S-1). In the presence of a biologically significant chelating agent such as serum albumin, a marked decrease in the Cu2aq-induced superoxide dismutation was observed. This was not the case when the dismutation in the presence of either the Cu2 of native erythrocuprein or Cu, Co-erythrocuprein, or those Cu2 ions chelated with tyrosine or certain di- and tripeptides was measured.     

Superoxide dismutase mimetic activity of cytokinin-copper(II) complexes

Dissociation constants of cytokinins, derivatives of purine which form complexes with cupric ion, were determined by spectrophotometry and the stability constants of their copper complexes by pH titration. The values found for kinetin were 3.76, 9.96, 7.8, and 15.3 for pK1, pK2, logk1, and log beta 2, respectively, and those for 6-benzylaminopurine were, in the same order, 3.90, 9.84, 8.3, and 15.9. The copper(II) complexes with kinetin and 6-benzylaminopurine had superoxide dismutase mimetic activity, and the reaction rate constants with superoxide, which were determined by polarography, were 2.3 X 10(-7) M-1 s-1 for kinetin and 1.5 X 10(-7) M-1 s-1 for 6-benzylaminopurine at pH 9.8 and 25 degrees C.     

EPR study of Cu(II) complexes of tridentate amino acids

The Cu(II) complexes of tridentate amino acids and related amines in alkaline solution were studied by EPR spectroscopy. Line shapes, g∥ and A∥ of each amino acid complex were compared with those of the corresponding amine complex. The results indicate that aromatic amino acids, monoaminodicarboxylic amino acids, arginine, methionine, and lysine bind to Cu(II) via the amino and carboxyl α groups. On the other hand cysteine, 2-3-diaminopropionic acid and hydroxy amino acids appear to be coordinated through the α-amino group and the third potentially binding group. Evidence is presented for the formation of mixed complexes in the cases of histidine and 2-4-diaminobutyric acid, whereas a glycine-like complex with apical coordination of the δ-amino groups is proposed for the ornithine-Cu(II) complex.     

Simulation of the diffusion-controlled reaction between superoxide and superoxide dismutase. II. Detailed models

A two-stage Brownian dynamics simulation method is used to study the diffusion-influenced bimolecular reaction between superoxide and superoxide dismutase (SOD). The crystal structure of the dimeric enzyme is used in constructing detailed topographical and electrostatic models. Several electrostatic models are considered. In the most realistic, the excluded volume of the protein, which is impermeable to penetration by mobile ions, is assigned a dielectric constant of 2 and the surrounding “solvent” is assigned a value of 78. A finite difference method is used to solve the linearized Poisson–Boltzmann equation. For native SOD, the simulations reproduce the pronounced salt dependence of the rate constant observed experimentally. This salt dependence is attributed to electrostatic interactions between enzyme and substrate that are inherently attractive and amplified by the low dielectric constant of the protein interior. The simulation method is also applied to a modified enzyme, acylated SOD.     

Cyanide binding to Cu, Zn superoxide dismutase. An NMR study of the Cu(II), Co(II) derivative

The Cu,Co superoxide dismutase derivative, in which the native Zn(II) was replaced by Co(II), was investigated by 1H NMR spectroscopy at pH 7.0 in the presence of CN- and N-3. Addition of either anion produced large but remarkably different variations in the position of the histidine proton signals bound to the metal cluster. The resonances of the histidines bound to the copper broadened at low CN- concentrations (6 X10(-5)-16.5 X 10(-3) M KCN, in the presence of 1.5 mM protein) and narrowed again, with changed chemical shifts at [KCN] greater than 10(-2) M. At 7 degrees C two resonances split into two pairs of lines as a function of [CN-]. The temperature dependence of these resonances, in the presence of nonsaturating [CN-], suggests a slow exchange between two forms of the protein-bound copper in the presence of the anion. The apparent activation parameters associated with the interconversion of the two species indicate a local conformational change in the presence of CN-. No evidence of temperature dependence was seen in the spectrum in the presence of N-3, which, on the other hand, was fully removed from the copper by addition of CN-. No evidence was obtained for removal by CN- of a histidine bound to the copper as previously reported for low affinity anions at pH 5.5 (Bertini, I., Lanini, G., Luchinat, C., Messori, L., Monanni, R., and Scozzafava, A. (1985) J. Am. Chem. Soc. 107, 4391-4396). These results indicate that CN- has a unique pattern of binding to the enzyme copper. Since catalytic and structural data indicate that CN- is the only appropriate substrate analogue for the Cu,Zn superoxide dismutase, data from anions with much less affinity may lead to misleading conclusions on the mechanism of anion and substrate binding to the enzyme.     

NMR studies on Cu(II)-peptide complexes: exchange kinetics and determination of structures in solution

The interaction of copper(II) with histidine containing peptides has recently acquired renewed interest following the established link between abnormal protein behaviour in neurodegenerative processes and unpaired copper homeostasis. Five peptide sequences taken from the amyloid precursor protein and the prion protein were considered. Addition of paramagnetic Cu(II) ions to solutions of such peptides was not found to severely affect the appearance of NMR spectra, thus limiting the usual approach for structural determination. Exchange kinetics was shown to play a major role in determining the observed paramagnetic spin-lattice relaxation rates. Two independent methods were suggested for evaluating the exchange rates of His-containing peptides from the copper-coordination sphere and to calculate copper-proton distances. In such a way NMR was demonstrated to have the potential of providing detailed structures of the Cu(II)-peptide complexes in solution.     

Development of novel copper(II) complexes of benzothiazole-N-sulfonamides as protective agents against superoxide anion. Crystal structures of [Cu(N–2-(4-methylbenzothiazole)benzenesulfonamidate)2(py)2] and [Cu(N–2-(6-nitrobenzothiazole)naphthalenesulfonamidate)2(py)2]

Copper(II) ternary complexes based on the novel benzothiazole- N-sulfonamides, HL1 ( N-2-(4-methylbenzothiazole)benzenesulfonamide) and HL2 ( N-2-(6-nitrobenzothiazole)naphthalenesulfonamide) ligands, and pyridine have been synthesized and characterized. Complexes [Cu(L1)(2)(py)(2)] (1). and [Cu(L2)(2)(py)(2)] (2). were chemically characterized and their structures determined by means of single crystal X-ray analysis. In both compounds the Cu(II) ion is coordinated to four N atoms in a nearly square planar arrangement. The Cu-N bond distances are similar to those of Cu(2)Zn(2)SOD. The SOD mimetic activity of the complexes was determined both in vitro and in vivo. For determining the SOD-like activity of the complexes in vivo, we have developed a new method based on the complexes' protective effect on a delta sod1mutant of Saccharomyces cerevisiae against free radicals generated by hydrogen peroxide and menadione as well as free radicals produced in the cellular respiration process. The results have shown that complex 1 presents a protective action against oxidative stress induced by menadione or H(2)O(2) and that both complexes 1 and 2 protect against free radicals generated in cellular respiration.