Mechanism of S-nitrosation of recombinant human brain calbindin D28K

Mass spectrometry and UV-vis absorption results support a mechanism for NO donation by S-nitrosoglutathione (GSNO) to recombinant human brain calbindin D(28K) (rHCaBP) that requires the presence of trace copper, added as either Cu,Zn-superoxide dismutase (CuZnSOD) or CuSO(4). The extent of copper-catalyzed rHCaBP S-nitrosation depends on the ratio of protein to GSNO and on the reaction time, and NO-transfer is prevented when copper chelators are present. CuZnSOD is an efficient catalyst of rHCaBP S-nitrosation, and the mechanism of CuZnSOD-catalyzed S-nitrosation involves reduction of the active-site Cu(II) by a number of the five free thiols in rHCaBP, giving rise to thiyl radicals. The Cu(I)ZnSOD formed catalyzes the reductive cleavage of GSNO present in solution to give GSH and release NO. rHCaBP thiyl radicals react with NO to yield the S-nitrosoprotein. Cu(II)ZnSOD is also reduced by GSH in a concentration-dependent manner up to 5 mM but not at higher GSH concentrations. However, unlike the rHCaBP thiyl radicals, GS(*) radicals dimerize to GSSG faster than their reaction with NO. The data presented here provide a biologically relevant mechanism for protein S-nitrosation by small S-nitrosothiols. S-nitrosation is rapidly gaining recognition as a major form of protein posttranslational modification, and the efficient S-nitrosation of CaBP by CuZnSOD/GSNO is speculated to be of neurochemical importance given that CaBP and CuZnSOD are abundant in neurons.     

Structure-reactivity studies of the Cu(2+)-catalyzed decomposition of four S-nitrosothiols based around the S-Nitrosocysteine/S-nitrosoglutathione structures.

S-Nitrosoglutathione (GSNO) and the dipeptide derivative S-nitrosoglutamylcysteine (SNO-GluCys) both at 1 x 10(-3) M in pH 7. 4 buffer containing added Cu(2+) (1 x 10(-5) M) are very unreactive toward decomposition (measured spectrophotometrically), and in both cases reaction stops at very low conversion. S-Nitrosocysteine (SNC) and the dipeptide derivative S-nitrosocysteinylglycine (SNO-CysGly), on the other hand, are orders of magnitude more reactive under the same conditions, and reaction proceeds to completion. Initially, we interpreted these results in terms of the requirement of a suitably positioned free NH(2) group (which is available in both SNC and SNO-CysGly, but not in GSNO and SNO-GluCys) for efficient complexation of Cu(+), the effective reagent. However, later results measured at much lower substrate concentration (1 x 10(-6) M) using the NO electrode system showed that at this concentration, all four S-nitrosothiols react at approximately the same rate and yield NO quantitatively. For GSNO the rate and percentage conversion were shown to drop progressively as the substrate concentration increases. All reactions are effectively halted in the presence of the metal ion chelator EDTA. The results can readily be explained in terms of complexation of Cu(2+) by the product disulfides from GSNO (i.e., GSSG) and SNO-GluCys, involving the glutamate residue, which is not present in SNC and SNO-CysGly. This is confirmed by the observed progressive reduction in yield and percentage conversion of GSNO decomposition as GSSG is added, at micromolar substrate concentrations.     

S-nitrosation of glutathione by nitric oxide,peroxynitrite, and (*)NO/O(2)(*-)

To elucidate potential mechanisms of S-nitrosothiol formation in vivo, we studied nitrosation of GSH and albumin by nitric oxide ((*)NO), peroxynitrite, and (*)NO/O(2)(*)(-). In the presence of O(2), (*)NO yielded 20% of S-nitrosoglutathione (GSNO) at pH 7.5. Ascorbate and the spin trap 4-hydroxy-[2,2,4,4-tetramethyl-piperidine-1-oxyl] (TEMPOL) inhibited GSNO formation by 67%. Electron paramagnetic resonance spectroscopy with 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) demonstrated intermediate formation of glutathionyl radicals, suggesting that GSNO formation by (*)NO/O(2) is predominantly mediated by (*)NO(2). Peroxynitrite-triggered GSNO formation (0.06% yield) was stimulated 10- and 2-fold by ascorbate and TEMPOL, respectively. Co-generation of (*)NO and O(2)(*)(-) at equal fluxes yielded less GSNO than (*)NO alone, but was 100-fold more efficient (8% yield) than peroxynitrite. Moreover, in contrast to the reaction of peroxynitrite, GSNO formation by (*)NO/O(2)(*)(-) was inhibited by ascorbate. Similar results were obtained with albumin instead of GSH. We propose that sulfhydryl compounds react with O(2)(*)(-) to initiate a chain reaction that forms radical intermediates which combine with (*)NO to yield GSNO. In RAW 264.7 macrophages, S-nitrosothiol formation by (*)NO/O(2) and (*)NO/O(2)(*)(-) occurred with relative efficiencies comparable to those in solution. Our results indicate that concerted generation of (*)NO and O(2)(*)(-) may essentially contribute to nitrosative stress in inflammatory diseases.     

Kinetics and mechanism of the decomposition of S-nitrosoglutathione by l-ascorbic acid and copper ions in aqueous solution to produce nitric oxide.

S-Nitrosothiols serve as a good source of nitric oxide ((*)NO) mainly due to the ease of cleavage of the S-N bond which consequently produces (*)NO. The reductive decomposition of S-nitrosoglutathione (GSNO) by l-ascorbic acid (vitamin C) yields (*)NO which was monitored both electrochemically (using NO-probe) and spectrophotometrically. The rate of reaction and (*)NO release was found to be pH dependent in a manner which drastically increases with pH demonstrating that the l-ascorbic acid dianion (A(2-)) is by far the most reactive species of l-ascorbic acid (H(2)A). The derived rate expression (measuring the disappearance of the absorption at ca. 336 nm due to GSNO) was established as rate = -d[GSNO](t)/dt = ((k(a)[H(+)](2) + k(b)[H(+)]K(1) + k(c)K(1)K(2))/([H(+)](2) + K(1)[H(+)] + K(1)K(2)))[GSNO](t)[H(2)A](t). k(a), k(b), and k(c) are second-order rate constants via the H(2)A, HA(-), and A(2-) pathways, respectively, while K(1) and K(2) represent the first and second equilibrium dissociation constants of l-ascorbic acid. There is little or no reaction at low pH (below 5.5), where H(2)A is a predominant species, and as a result the rate constant (k(a)) via this route was found to be negligible. At 25 degrees C, k(b) = 5.23 +/- 1.47 x 10(-3) dm(3) mol(-1) s(-1) and k(c) = 1.22 +/- 0.04 x 10(3) dm(3) mol(-1) s(-1), activation parameters DeltaH(double dagger)(b) = 54.4 +/- 4.3 kJ mol(-1), DeltaS(double dagger)(b) = -106 +/- 16 J K(-1) mol(-1), DeltaH(double dagger)(c) = 80.5 +/- 7.5 kJ mol(-1), DeltaS(double dagger)(c) = 84 +/- 7 kJ mol(-1). The experimental rate and activation parameters suggest that this redox process follows an outer-sphere electron transfer mechanism. GSNO is relatively stable in the dark, aqueous medium and even in the presence of trace quantities of Cu(2+). Induced catalytic decomposition of GSNO only becomes significant above ca. 10 microM Cu(2+), but after this it shows linear dependency. To nullify any catalysis by Cu(2+) or any other transition metal ions, EDTA was added to all experimental reactions except those where catalysis by Cu(2+) was studied.     

S-nitrosation controls gating and conductance of the alpha 1 subunit of class C L-type Ca(2+) channels

Modulation of smooth muscle, L-type Ca(2+) channels (class C, Ca(V)1.2b) by thionitrite S-nitrosoglutathione (GSNO) was investigated in the human embryonic kidney 293 expression system at the level of whole-cell and single-channel currents. Extracellular administration of GSNO (2 mM) rapidly reduced whole-cell Ba(2+) currents through channels derived either by expression of alpha1C-b or by coexpression of alpha1C-b plus beta2a and alpha2-delta. The non-thiol nitric oxide (NO) donors 2,2-diethyl-1-nitroso-oxhydrazin (2 mM) and 3-morpholinosydnonimine-hydrochloride (2 mM), which elevated cellular cGMP levels to a similar extent as GSNO, failed to affect Ba(2+) currents significantly. Intracellular administration of copper ions, which promote decomposition of the thionitrite, antagonized its inhibitory effect, and loading of cells with high concentrations of dithiothreitol (2 mM) prevented the effect of GSNO on alpha1C-b channels. Intracellular loading of cells with oxidized glutathione (2 mM) affected neither alpha1C-b channel function nor their modulation by GSNO. Analysis of single-channel behavior revealed that GSNO inhibited Ca(2+) channels mainly by reducing open probability. The development of GSNO-induced inhibition was associated with the transient occurrence of a reduced conductance state of the channel. Our results demonstrate that GSNO modulates the alpha1 subunit of smooth muscle L-type Ca(2+) channels by an intracellular mechanism that is independent of NO release and stimulation of guanylyl cyclase. We suggest S-nitrosation of intracellularly located sulfhydryl groups as an important determinant of Ca(2+) channel gating and conductance.     

Thiol-copper(I) and disulfide-dicopper(I) complex O2-reactivity leading to sulfonate-copper(II) complex or the formation of a cross-linked thioether-phenol product with phenol addition

In order to better understand copper mediated oxidative chemistry via ligand-Cu(I)/O(2) reactivity employing S-donor ligands for copper, O(2)-reactivity studies of the copper(I) complexes (1 and 2, Chart 2) have been carried out with a tridentate N(2)S thiol ligand (1-(N-methyl-N-(2-(pyridin-2-yl)ethyl)amino)propane-2-thiol; L(SH)) or its oxidized disulfide form (L(SS)). Reactions of [L(SH)Cu(I)](+) (1) and [L(SS)(Cu(I))(2)(X)(2)](2+) (2) with O(2) give approximately 90% and approximately 70% yields of [L(SO3)Cu(II)(MeOH)(2)](+) (3), respectively, where L(SO3) is S-oxygenated sulfonate; 3 was characterized by electrospray ionization (ESI) mass spectrometry and X-ray crystallography. Mimicking TyrCys galactose oxidase cofactor biogenesis, a new C-S bond is formed (within new thioether moiety L(SPhOH)) from cuprous complex (both 1 and 2) dioxygen reactivity in the presence of 2,4-tBu(2)-phenolate. In addition, the disulfide ligand (L(SS)) reacts with 2equiv. cupric ion salts and the phenolate to efficiently give the cross-linked product L(SPhOH) in high yield (>90%) under anaerobic conditions. Separately, complex [L(SPhO)Cu(II)(ClO(4))] (4), possessing the cross-linked L(SPhOH), was characterized by ESI mass spectrometry and X-ray crystallography.     

Heme nitrosylation of deoxyhemoglobin by s-nitrosoglutathione requires copper

NO reactions with hemoglobin (Hb) likely play a role in blood pressure regulation. For example, NO exchange between Hb and S-nitrosoglutathione (GSNO) has been reported in vitro. Here we examine the reaction between GSNO and deoxyHb (HbFe(II)) in the presence of both Cu(I) (2,9-dimethyl-1, 10-phenanthroline (neocuproine)) and Cu(II) (diethylenetriamine-N,N,N',N",N"-pentaacetic acid) chelators using a copper-depleted Hb solution. Spectroscopic analysis of deoxyHb (HbFe(II))/GSNO incubates shows prompt formation (<5 min) of approximately 100% heme-nitrosylated Hb (HbFe(II)NO) in the absence of chelators, 46% in the presence of diethylenetriamine-N,N,N',N",N"-pentaacetic acid, and 25% in the presence of neocuproine. Negligible (<2%) HbFe(II)NO was detected when neocuproine was added to copper-depleted HbFe(II)/GSNO incubates. Thus, HbFe(II)NO formation via a mechanism involving free NO generated by Cu(I) catalysis of GSNO breakdown is proposed. GSH is a source of reducing equivalents because extensive GSSG was detected in HbFe(II)/GSNO incubates in the absence of metal chelators. No S-nitrosation of HbFe(II) was detected under any conditions. In contrast, the NO released from GSNO is directed to Cysbeta(93) of oxyHb in the absence of chelators, but only metHb formation is observed in the presence of chelators. Our findings reveal that the reactions of GSNO and Hb are controlled by copper and that metal chelators do not fully inhibit NO release from GSNO in Hb-containing solutions.     

Copper-release from yeast Cu(I)-metallothionein by nitric oxide (NO)

The reaction of yeast Cu-MT with nitric oxide (NO) was examined. A release of copper from the Cu(I)-thiolate clusters of the protein by this remarkably important reagent was observed in vitro. The characteristic spectroscopic signals of the Cu(I)-thiolate chromophores levelled off in the presence of a two-fold molar excess of NO expressed per equivalent of thionein-copper as monitored by UV-electronic absorption, circular dichroism and luminescence emission. At the same time all of the copper became EPR detectable. The oxidized metal ions could easily be removed from the protein moiety by gelfiltration. The reversibility of the copper releasing process is of special interest. The specific fluorescence and dichroic properties of the previously demetallized protein could be recovered up to 85% under reductive conditions. Moreover, no difference in the electrophoretic behaviour was seen compared to the untreated Cu-MT. Thus, NO may act as a potent metabolic source for the transient copper release from Cu-MT. In the course of an oxidative burst this highly Fenton active copper is able to improve the efficacy of biological defence mechanisms.     

Nitrosylation of manganese(II) tetrakis(N-ethylpyridinium-2-yl)porphyrin: a simple and sensitive spectrophotometric assay for nitric oxide.

Reaction between NO(*) and manganese tetrakis(N-ethylpyridinium-2-yl)porphyrin (Mn(III)TE-2-PyP(5+)) was investigated at 25 degrees C. At high excess of NO(*) (1.5 mM) the reaction with the oxidized, air-stable form Mn(III)TE-2-PyP(5+) (5 microM), proceeds very slowly (t(1/2) congruent with 60 min). The presence of excess ascorbate (1 mM) produces the reduced form, Mn(II)TE-2-PyP(4+), which reacts with NO(*) stoichiometrically and in the time of mixing (k congruent with 1 x 10(6) M(-1) s(-1)). The high rate of formation and the stability of the product, Mn(II)TE-2-PyP(NO)(4+) (?Mn(NO)?(6)), make the reaction outcompete the reaction of NO(*) with O(2). Our in vitro measurements show a linear absorbance response upon addition of NO to a PBS, pH 7.4, solution containing an excess of ascorbate over Mn(III)TE-2-PyP(5+). Thus, the observed interactions can be the basis of a convenient and sensitive spectrophotometric assay for NO(*). Also, it may have important implications for the in vivo behavior of Mn(III)TE-2-PyP(5+) which is currently exploited as a possible therapeutic agent for various oxygen-radical related disorders.     

Molecular structure, bioavailability and bioactivity of [Cu(o-phen)2(cnge)](NO3)2.2H2O and [Cu(o-phen)(cnge)(H2O)(NO3)2] complexes

Two Cu(II) complexes with cyanoguanidine (cnge) and o-phenanthroline, [Cu(o-phen)(2)(cnge)](NO(3))(2).2H(2)O (1) and [Cu(o-phen)(cnge)(H(2)O)(NO(3))(2)] (2), have been synthesized using different experimental techniques and characterized by elemental analyses, FTIR, diffuse and UV-vis spectra and EPR and magnetic moment measurements techniques. The crystal structures of both complexes were solved by X-ray diffraction methods. Complex (1) crystallizes in the monoclinic space group C2/c with a=12.621(5), b=31.968(3), c=15.39(1)A, beta=111.68(4) degrees, and Z=8 and complex (2) in the monoclinic space group P2(1)/n with a=10.245(1), b=13.923(2), c=12.391(2)A, beta=98.07(1) degrees, and Z=4. The environments of the copper(II) center are trigonal bipyramidal (TBP) for [Cu(o-phen)(2)(cnge)](2+) and an elongated octahedron for [Cu(o-phen)(cnge)(H(2)O)(NO(3))(2)]. Solution studies have been performed to determine the species distribution. The superoxide dismutase (SOD) activities of both complexes have also been tested in order to determine if these compounds mimic the enzymatic action of the enzyme SOD that protects cells against peroxide radicals.     

Nitric oxide inhibits depolarization-evoked glutamate release from rat cerebellar granule cells.

Nitric oxide (NO) modulates the release of various neurotransmitters, some of these are considered to be involved in neuronal plasticity that includes long-term depression in the cerebellum. To date, there have been no reports on the modulation of the exocytotic release of neurotransmitters in the cerebellar granule cells (CGCs) by NO. The aim of this study was to investigate the effects of NO on the exocytotic release of glutamate from rat CGCs. Treatment with NO-related reagents revealed that NO inhibited high-K(+)-evoked glutamate release. Clostridium botulinum type B neurotoxin (BoNT/B) attenuated the enhancement of glutamate release caused by NO synthase (NOS) inhibition; this indicates that NO acts on the high-K(+)-evoked exocytotic pathway. cGMP-related reagents did not affect the high-K(+)-evoked glutamate release. NO-related reagents did not affect Ca(2+) ionophore-induced glutamate release, suggesting that NO inhibits Ca(2+) entry through voltage-dependent Ca(2+) channels (VDCC). Monitoring of intracellular Ca(2+) revealed that NO inhibited high-K(+)-evoked Ca(2+) entry. L-type VDCC blockers inhibited glutamate release and NO did not have an additive effect on the inhibition produced by the L-type VDCC blocker. The inhibition of the high-K(+)-evoked glutamate release by NO was abolished by a reducing reagent; this suggested that NO regulates the high-K(+)-evoked glutamate release from CGCs by redox modulation.     

Synthesis,crystal structure,and nuclease activity of planar mono-heterocyclic base copper(II) complexes

A series of mononuclear copper(II) complexes having a 1:1 molar ratio of copper and the planar heterocyclic base like 1,10-phenanthroline (phen), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq) and dipyrido[3,2-a:2',3'-c]phenazine (dppz) are prepared from a reaction of copper(II) nitrate.trihydrate and the base (L) in ethanol or aqueous ethanol at different temperatures. The complexes [Cu(dpq)(NO(3))(2)] (2), [Cu(dpq)(NO(3))(H(2)O)(2)](NO(3)) (3), [Cu(dpq)(NO(3))(2)(H(2)O)(2)].2H(2)O (4.2H(2)O) and [Cu(dppz)(NO(3))(2)(H(2)O)].H(2)O (5.H(2)O) have been characterized by X-ray crystallography. The crystal structures show the presence of the heterocyclic base in the basal plane. The coordination geometries of the copper(II) centers are axially elongated square-pyramidal (4+1) in 2, 3 and 5, and octahedral (4+2) in 4. The nitrate anion in the coordination sphere displays unidentate and bidentate chelating bonding modes. The axial ligand is either H(2)O or NO(3) in these structures giving a Cu-L(ax) distance of approximately 2.4 A. The one-electron paramagnetic complexes (mu approximately 1.8 mu(B)) exhibit axial EPR spectra in DMF glass at 77 K giving g(parallel)>g( perpendicular ) with an A(parallel) value of approximately 170G indicating a [d(x)2(-y)2](1) ground state. The complexes are redox active and display a quasireversible cyclic voltammetric response for the Cu(II)/Cu(I) couple near 0.0 V vs. SCE giving an order of the E(1/2) values as 5(dppz)>2-4 (dpq)>[Cu(phen)(2)(H(2)O)](2+)>1 (phen). The complexes bind to calf thymus DNA giving an order 5 (dppz)>2 (dpq)>[Cu(phen)(2)(H(2)O)](2+)>1 (phen). An effect of the extended planar ring in dpq and dppz is observed in the DNA binding. The complexes show nuclease activity with pUC19 supercoiled DNA in DMF/Tris-HCl buffer containing NaCl in presence of mercaptopropanoic acid as a reducing agent. The extent of cleavage follows the order: [Cu(phen)(2)(H(2)O)](ClO(4))(2)>5>2 approximately 3 approximately 4>1. The bis-phen complex is a better cleaver of SC DNA than 1-5 having mono-heterocyclic base. Mechanistic investigations using distamycin reveal minor groove biding for the phen, dpq complexes, and a major groove binding for the dppz complex 5. The cleavage reactions are found to be inhibited in the presence of hydroxyl radical scavenger DMSO and the reactions are proposed to proceed via sugar hydrogen abstraction pathway. The ancillary ligand is found to have less effect in DNA binding but are of importance in DNA cleavage reactions.     

Iron(III)- and copper(II) complexes of an asymmetric, pentadentate salen-like ligand bearing a pendant carboxylate group

The equilibrium and solution structural properties of the iron(III) and copper(II) complexes of an asymmetric salen-like ligand (N,N'-bis(2-hydroxybenzyl)-2,3-diamino-propionic acid, H(3)bhbdpa) bearing a pendant carboxylate group were characterized in aqueous solution by potentiometric, pH-dependent electron paramagnetic resonance (EPR) and UV-Vis (UV-Visible) measurements. In the equimolar systems the pentadentate ligand forms very stable, differently protonated mononuclear complexes with both metal ions. In the presence of iron(III) {NH, PhO(-), COO(-)}, {2NH, 2PhO(-), COO(-)} and {2NH, 2PhO(-), COO(-), OH(-)} coordinated complexes are dominant. The EPR titrations reflected the presence of microscopic complex formation pathways, leading to the formation of binding isomers in case of Cu(H(2)bhbdpa)(+), Cu(Hbhbdpa) and Cu(bhbdpa)(-). The {2NH, 2PhO(-)+COO(-)/H(2)O} coordinated Cu(bhbdpa) is the only species between pH 6-11. At twofold excess of metal ion dinuclear complexes were detected with both iron(III) and copper(II). In presence of iron(III) a mu-carboxylato-mu-hydroxo-bridged dinuclear complex (Fe(2)(bhbdpa)(OH)(3)) is formed from Fe(H(2)bhbdpa)(2+) through overlapping proton release processes, providing one of the rare examples for the stabilization of an endogenous carboxylate bridged diiron core in aqueous solution. The complex Cu(2)(bhbdpa)(+) detected in the presence of copper(II) is a paramagnetic (S=1) species with relatively weakly coupled metal ions.     

Further evidence supporting an inner sphere mechanism in the NO reduction of the copper(II) complex Cu(dmp)2 (dmp = 2,9-dimethyl-1,10-phenanthroline)

Described are further studies directed towards elucidating the mechanism of the nitric oxide reduction of the copper(II) model system, Cu(dmp)2(2+) (I, dmp=2,9-dimethyl-1,10-phenanthroline). The reaction of I with NO in methanol results in the formation of Cu(dmp)2+ (II) and methyl nitrite (CH3ONO), with a second order rate constant kNO=38.1 M-1 s-1 (298K). The activation parameters for this reaction in buffered aqueous medium were measured to be DeltaH(double dagger)=41.6 kJ/mol and DeltaS(double dagger)=-82.7 kJ/mol deg. The addition of azide ion (N3-) as a competing nucleophile results in a marked acceleration in the rate of the copper(II) reduction. Analysis of the kinetics for the NO reduction of the bulkier Cu(dpp)(2)2+ (IV, dpp=2,9-diphenyl-1,10-phenanthroline) and the stronger oxidant, Cu(NO2-dmp)2(2+) (V, NO2-dmp=5-nitro-2,9-dimethyl-1,10-phenanthroline), gave the second order rate constants kNO=21.2 and 29.3 M-1 s-1, respectively. These results argue against an outer sphere electron transfer pathway and support a mechanism where the first step involves the formation of a copper-nitrosyl (Cu(II)-NO or Cu(I)-NO+) adduct. This would be followed by the nucleophilic attack on the bound NO and the labilization of RONO to form the nitrite products and the cuprous complex.     

Measurement of antioxidant ability of melatonin and serotonin by the DMPD and CUPRAC methods as trolox equivalent

Melatonin (N-acetyl-5-methoxytryptamine) is the chief secretory product of the pineal gland and synthesized enzymatically from serotonin (5-hydroxytryptamine). These indoleamine derivatives play an important role in the prevention of oxidative damage. In the present study, DMPD radical scavenging and cupric ion (Cu(2+)) reducing ability of melatonin and serotonin as trolox equivalent antioxidant activity (TEAC) was investigated. Melatonin and serotonin demonstrated 73.5 and 127.4 microg/mL trolox equivalent DMPD( radical+) scavenging activity at the concentration of 100 microg/mL. Also, at the same concentration, melatonin and serotonin showed 14.41 and 116.09 microg/mL trolox equivalent cupric ion (Cu(2+)) reducing ability. These results showed that melatonin and serotonin had marked DMPD(radical+) radical scavenging and cupric ions (Cu(2+)) reducing ability. Especially, serotonin had higher DMPD radical scavenging and cupric ions (Cu(2+)) reducing activity than melatonin because of its phenolic group.     

A manganese superoxide dismutase (MnSOD) from Ruditapes philippinarum: comparative structural- and expressional-analysis with copper/zinc superoxide dismutase (Cu/ZnSOD) and biochemical analysis of its antioxidant activities

Superoxide dismutases (SODs), antioxidant metalloenzymes, represent the first line of defense in biological systems against oxidative stress caused by excessive reactive oxygen species (ROS), in particular O(2)(?-). Two distinct members of SOD family were identified from Manila clam Ruditapes philippinarum (abbreviated as RpMnSOD and RpCu/ZnSOD). The structural analysis revealed all common characteristics of SOD family in both RpSODs from primary to tertiary levels, including three MnSOD signatures and two Cu/ZnSOD signatures as well as invariant Mn(2+)- and Cu/Zn(2+)-binding sites in RpMnSOD and RpCu/ZnSOD, respectively. Putative RpMnSOD and RpCu/ZnSOD proteins were predicted to be localized in mitochondrial matrix and cytosol, respectively. They shared 65.2% and 63.9% of identity with human MnSOD and Cu/ZnSOD, respectively. Phylogentic evidences indicated the emergence of RpSODs within molluscan monophyletic clade. The analogous spatial expression profiles of RpSODs demonstrated their higher mRNA levels in hemocytes and gills. The experimental challenges with poly I:C, lipopolysaccharide and Vibrio tapetis illustrated the time-dependent dynamic expression of RpSODs in hemocytes and gills. The recombinant RpMnSOD was expressed in a prokaryotic system and its antioxidant property was studied. The rRpMnSOD exhibited its optimum activity at 20?°C, under alkaline condition (pH 9) with a specific activity of 3299?U?mg(-1). These outcomes suggested that RpSODs were constitutively expressing inducible proteins that might play crucial role(s) in innate immunity of Manila clam.     

Pt(II) complexes with N-(3-pyridyl)-2-(4-(trifluoromethyl)phenyl)diazenecarboxamide and their reactions with glutathione

The reaction between [PtCl(dmso)(en)]Cl (dmso=dimethyl sulfoxide, en=ethylenediamine) and N-(3-pyridyl)-2-(4-(trifluoromethyl)phenyl)diazenecarboxamide (L) was studied using multinuclear NMR spectroscopy. The water-soluble complexes [PtCl(en)(L-N1)](+) (1) and [Pt(en)(L-N1)(2)](2+) (2) were isolated and their reactions with glutathione (GSH) were investigated to assess the oxidation properties of coordinated L. Both species 1 and 2 oxidized GSH to GSSG, while the reduced form of L (semicarbazide, SL) remained coordinated to Pt(2+). In complex 1 the labile chloride ion was substituted by the thiol moiety of GSH, which gave rise to the release of en in excess GSH over a period of 7 days. Complexes [PtCl(dmso)(en)]Cl, 1, 2 and ligand L were tested against T24 bladder carcinoma cells. Ligand L and complexes 1 and 2 showed higher cytotoxicity than [PtCl(dmso)(en)]Cl.     

Characterization of the metal-substituted dipeptidyl peptidase III (rat liver)

Dipeptidyl peptidase III (DPP III) (EC 3.4.14.4), which has a HELLGH-E (residues 450-455, 508) motif as the zinc binding site, is classified as a zinc metallopeptidase. The zinc dissociation constants of the wild type, Leu(453)-deleted, and E508D mutant of DPP III at pH 7.4 were 4.5 (+/-0.7) x 10(-13), 5.8 (+/-0.7) x 10(-12), and 3.2 (+/-0.9) x 10(-10) M, respectively. The recoveries of the enzyme activities by the addition of various metal ions to apo-DPP III were also measured, and Co(2+), Ni(2+), and Cu(2+) ions completely recovered the enzyme activities as did Zn(2+). The dissociation constants of Co(2+), Ni(2+), and Cu(2+) ions for apo-DPP III at pH 7.4 were 8.2 (+/-0.9) x 10(-13), 2.7 (+/-0.3) x 10(-12), and 1.1 (+/-0.1) x 10(-14) M, respectively. The shape of the absorption spectrum of Co(2+)-DPP III was very similar to that of Co(2+)-carboxypeptidase A or Co(2+)-thermolysin, in which the Co(2+) is bound to two histidyl nitrogens, a water molecule, and a glutamate residue. The absorption spectrum of Cu(2+)-DPP III is also very similar to that of Cu(2+)-thermolysin. The EPR spectrum and the EPR parameters of Cu(2+)-DPP III were very similar to those of Cu(2+)-thermolysin but slightly different from those of Cu(2+)-carboxypeptidase A. The five lines of the superfine structure in the perpendicular region of the EPR spectrum in Cu(2+)-DPP III suggest that nitrogen atoms should coordinate to the cupric ion in Cu(2+)-DPP III. All of these data suggest that the donor set and the coordination geometry of the metal ions in DPP III, which has the HExxxH motif as the metal binding site, are very similar to those of the metal ions in thermolysin, which has the HExxH motif.     

In vivo anticancer, anti-inflammatory, and toxicity studies of mixed-ligand Cu(II) complexes of dien and its Schiff dibases with heterocyclic aldehydes and 2-amino-2-thiazoline. Crystal structure of [Cu(dien)(Br)(2a-2tzn)](Br)(H(2)O)

A new series of complexes of the type [Cu(dien)(2a-2tzn)Y(2)] and [Cu(dienXX)(2a-2tzn)Y(2)], where dien=diethylenetriamine and dienXX=Schiff dibase of diethylenetriamine formed with 2-furaldehyde (dienOO), 2-thiophenecarboxaldehyde (dienSS), or pyrrol-2-carboxaldehyde (dienNN); Y=Cl, Br or NO(3); and 2a-2tzn=2-amino-2-thiazoline, were synthesized and their structure established by C, H, N and Cu analysis; IR and electronic spectra; magnetic susceptibility; and molar conductivity. The isolated complexes are monomers, paramagnetic, and electrolytes of types 1:1 or 1:2. In both types of solid state complexes, [Cu(dien)(2a-2tzn)Y(2)] and [Cu(dienXX)(2a-2tzn)Y(2)], dien and its Schiff dibases are bonded to Cu(II) in a tridentate fashion through 3N atoms. The coordination sphere is completed by the endocyclic nitrogen of the thiazoline moiety and by two Cl, Br, or NO(3) groups with distorted octahedral geometry. The proposed structure of these compounds was supported by X-ray analysis of [Cu(dien)(Br)(2a-2tzn)](Br)(H(2)O). The coordination polyhedron around the copper atom can be described as a distorted square pyramid [Cu(dien)(Br)(2a-2tzn)](+). Its basal plane is occupied by the four nitrogen atoms of the dien and thiazoline ligands with Cu-N distances ranging between 1.996(6) and 2.032(3)A, and the axial position is occupied by one of the two bromine atoms (Br1) with a Cu1-Br1 bond distance of 2.782(1)A. The second bromine atom (Br2) is 4.694(2)A from the copper atom, which exists as a discrete anion and is responsible for the cationic nature of the complex. Results regarding toxicity, antitumor, and anti-inflammatory activities of the investigated compounds are promising and allow the selection of a lead compound for further biological studies.     

The binding of copper(II) and zinc(II) to oxidized glutathione

1H and 13C NMR studies of Zn(II) binding to oxidized glutathione (GSSG) in aqueous solution over the pH range 4-11 show that it forms a complex with a 1:1 Zn:GSSG stoichiometry. At pH values between 6 and 11 the metal ligands are the COO- and NH2 groups of the glutamate residues. Below pH 5 the glycine end of the molecule also binds to the metal ions. EPR and visible absorption spectra of Cu(II) GSSG solutions suggest that similar complexes are formed with Cu(II). The solid products obtained from these solutions are shown by analysis and EPR to be primarily binuclear with Cu2GSSG stoichiometry, although the structures depend on the pH and stoichiometry of the solution from which they were obtained.