Direct evidence for inhibition of free radical formation from Cu(I) and hydrogen peroxide by glutathione and other potential ligands using the EPR spin-trapping technique.

Copper-induced oxidative damage is generally attributed to the formation of the highly reactive hydroxyl radical by a mechanism analogous to the Haber-Weiss cycle for Fe(II) and H2O2. In the present work, the reaction between the Cu(I) ion and H2O2 is studied using the EPR spin-trapping technique. The hydroxyl radical adduct was observed when Cu(I), dissolved in acetonitrile under N2, was added to pH 7.4 phosphate buffer containing 100 mM 5,5-dimethyl-1-pyrroline N-oxide (DMPO). Formation of the hydroxyl radical was dependent on the presence of O2 and subsequent formation of H2O2. The kscav/kDMPO ratios obtained were below those expected for a mechanism involving free hydroxyl radical and reflect the interference of nucleophilic addition of H2O to DMPO to form the DMPO/.OH adduct in the presence of nonchelated copper ion. Addition of ethanol or dimethyl sulfoxide to the reaction suggests that a high-valent metal intermediate, possibly Cu(III), was also formed. Spin trapping of hydroxyl radical was almost completely inhibited upon addition of Cu(I) to a solution of either nitrilotriacetate or histidine, even though the copper was fully oxidized to Cu(II) and H2O2 was formed. Bathocuproinedisulfonate, thiourea, and reduced glutathione all stabilized the Cu(I) ion toward oxidation by O2. Upon addition of H2O2, the Cu(I) in all three complexes was oxidized to varying degrees; however, only the thiourea complex was fully oxidized within 2 min of reaction and produced detectable hydroxyl radicals. No radicals were detected from the bathocuproinedisulfonate or glutathione complexes. Overall, these results suggest that the deleterious effects of copper ions in vivo are diminished by biochemical chelators, especially glutathione, which probably has a major role in moderating the toxicological effects of copper.     

Mechanistic flexibility in the reduction of copper(II) complexes of aliphatic polyamines by mercapto amino acids

The reactions of copper(II)-aliphatic polyamine complexes with cysteine, cysteine methyl ester, penicillamine, and glutathione have been investigated, with the goal of understanding the relationship between RS- -Cu(II) adduct structure and preferred redox decay pathway. Considerable mechanistic flexibility exists within this class of mercapto amino acid oxidations, as changes in the rate law could be induced by modest variations in reductant concentration (at fixed [Cu(II)]0), pH, and the structure of the redox partners. With excess cysteine present at 25 degrees C, pH 5.0, I = 0.2 M (NaOAc), decay of 1:1 cys-S- -Cu(II) transient adducts was found to be first order in both cys-SH and transient. Second-order rate constants characteristic of Cu(dien)2+(6.1 X 10(3) M-1 sec-1), Cu(Me5dien)2+ (2.7 X 10(3) M-1 sec-1), Cu(en)22+ (2.1 X 10(3) M-1 sec-1), and Cu(dien)22+ (4.7 X 10(3) M-1 sec-1) are remarkably similar, considering substantial differences in the composition and geometry of the oxidant first coordination sphere. A mechanism involving attack of cysteine on the coordinated sulfur atom of the transient, giving a disulfide anion radical intermediate, is proposed to account for these results. Moderate reactivity decreases in the cysteine-Cu(dien)2+, Cu(Me5dien)2+ reactions with increasing [H+] (pH 4-6) reflect partial protonation of the polyamine ligands. A very different rate law, second order in the RS- -Cu(II) transient and approximately zeroth order in mercaptan, applies in the pH 5.0 oxidations of cysteine methyl ester, penicillamine, and glutathione by Cu(dien)2+ and Cu(Me5dien)2+. This behavior suggests the intermediacy of di-mu-mercapto-bridged binuclear Cu(II) species, in which a concerted two-electron change yields the disulfide and Cu(I) products. Similar hydroxo-bridged intermediates are proposed to account for the transition from first- to second-order transient dependence in cysteine oxidations by Cu(dien)2+ and Cu(Me5dien)2+ as the pH is increased from 5 to 7. Yet another rate law, second order in transient and first order in cysteine, applies in the pH 5.0 oxidation of cysteine by Cu(Me6tren)2+ (k(25 degrees C) 7.5 X 10(7) M-2 sec-1, I = 0.2M). Steric rigidity of this trigonal bipyramidal oxidant evidently protects the coordinated sulfur atom from attack in a RSSR- -forming pathway. Formation of a coordinated disulfide in the rate-determining step is proposed, coupled with attack of a noncoordinated cysteine molecule on a vacated coordination position to stabilize the (Me6tren)Cu(I) product.     

Oxidation-reduction reactions in Ehrlich cells treated with copper-neocuproine.

The interaction of 2,9-dimethyl-1,10-phenanthroline (neocuproine or NC) and its copper complex with Ehrlich ascites tumor cells was studied. NC is frequently used as a negative control in studies of in vitro DNA degradation by copper phenanthroline and has also found use as a potential inhibitor of damage from oxidative stress in biological systems. NC inhibited Ehrlich cell growth in monolayer culture over 48 h treatment by 50% at 0.05 nmol/10(5) cells. Addition of 5- to 100-fold ratios of CuCl2 to NC (at 0.035 nmol NC/10(5) cells) produced progressively more growth inhibition. Addition of 1:0.5 ratios of NC to CuCl2 over the range of NC concentrations 0.08-0.2 nmol/10(5) cells/mL resulted in DNA single-strand breakage during 1-h treatments as measured by DNA alkaline elution. Concomitant addition of catalase or dimethyl sulfoxide (DMSO) inhibited DNA strand scission, while superoxide dismutase enhanced breakage. Catalase and DMSO also inhibited induction of membrane permeability by the copper complex of NC. These cellular effects apparently result from the intracellular generation of hydroxyl radical from H2O2. NC facilitated the uptake of copper into cells, though it was initially bound as a copper-histidine-like complex. The internalized copper was reduced to Cu(I), bound mostly as (NC)2Cu(I). To explain the (NC)2Cu-dependent generation of hydroxyl radical, it is hypothesized that glutathione successfully competes for Cu(I), converting it to a redox-active form that can catalyze the reduction of molecular oxygen to .OH. Model studies support this view. Radical scavengers did not reverse growth inhibition produced by NC or NC + CuCl2.     

The formation and nature of the mixed valence copper-D-penicillamine-chloride cluster in aqueous solution and its relevance to the treatment of Wilson's disease

Complex formation between D-penicillamine (Pen) and copper(II) ions has been studied under simulated physiological conditions in both the presence and absence of the blood plasma constituents albumin, alanine, histidine, and zinc(II). Chromatographic and uv/vis and electron spin resonance (esr) spectroscopic methods were used. The major species formed, at neutral pH and 0.15 mol dm-3 NaCl, is the violet species which is shown to have the same stoichiometry as the recently reported solid-state complex, i.e., [Cu8I Cu6II (Pen)12 Cl] 5-. The rate of formation of this species (MVC) is shown to be dependent on the Cu concentration, Cu:Pen ratio, relative Cl- ion concentration, pH, and temperature. Formation is inhibited by the presence of O2 and biological chelates. At the concentration levels found in blood plasma it is unlikely that the MVC ion has any significance in the therapeutic action of penicillamine in the treatment of Wilson's disease. Reexamination of the aqueous Cu-albumin-pen system reinforces earlier findings that pen is unable to mobilize Cu that is bound to albumin. Significant binding of pen to the protein is observed is not related to any protein-bound copper ions. Evidence that ternary complexes of the type amino acid-Cu-Pen can form in blood plasma is presented. These are unlikely, however, to be physiologically significant and the copper depletion induced by Pen in Wilson's disease cases must be elsewhere than in the blood plasma compartment.     

Structural Snapshots from the Oxidative Half-reaction of a Copper Amine Oxidase: IMPLICATIONS FOR O2 ACTIVATION*

The mechanism of molecular oxygen activation is the subject of controversy in the copper amine oxidase family. At their active sites, copper amine oxidases contain both a mononuclear copper ion and a protein-derived quinone cofactor. Proposals have been made for the activation of molecular oxygen via both a Cu(II)-aminoquinol catalytic intermediate and a Cu(I)-semiquinone intermediate. Using protein crystallographic freeze-trapping methods under low oxygen conditions combined with single-crystal microspectrophotometry, we have determined structures corresponding to the iminoquinone and semiquinone forms of the enzyme. Methylamine reduction at acidic or neutral pH has revealed protonated and deprotonated forms of the iminoquinone that are accompanied by a bound oxygen species that is likely hydrogen peroxide. However, methylamine reduction at pH 8.5 has revealed a copper-ligated cofactor proposed to be the semiquinone form. A copper-ligated orientation, be it the sole identity of the semiquinone or not, blocks the oxygen-binding site, suggesting that accessibility of Cu(I) may be the basis of partitioning O₂ activation between the aminoquinol and Cu(I).     

Mononuclear and polynuclear copper complexes of some substituted hydrazones

The reactivity of a series of potentially tetradentate hydrazone ligands, involving pyridyl and imidazolyl substituent groups, towards copper(II) salts has been examined. Both mononuclear and polynuclear derivatives are obtained with some ligands and in some cases redox reactions are observed in which, when water is a significant solvent component, nitrogen gas evolution indicates the formation of copper(I) derivatives. The reduction is assumed to occur by initial hydrolysis of the hydrazone ligand, forming hydrazine as one product, which reduces copper(II) to copper(I). However the copper(I) ions bind preferentially to unoxidized ligand thus limiting the extent to which reduction occurs. In the presence of electronegative ligands the copper(II) complexes are stabilized in some cases, while in one case a mixed valence polynuclear species is produced. Preliminary details of the X-ray structure of [Cu(IMAA)Br₂]·H₂O (IMAA = (1-methyl-2-imidazolyl)aldazine) indicate a mononuclear, five-coordinate, system involving unsymmetrical tridentate ligand, a structural feature which is apparent in most other mononuclear species.     

Penicillamine deprotonations and interactions with copper ions

In aqueous solutions about five times as many penicillamine molecules possess ionized sulfhydryl groups as deprotonated amino groups. That the corresponding ratio for cysteine is two is reaffirmed. N-Acetyl-dl-penicillamine and d-penicillamine react with Cu(II), even in acid solutions, to yield the corresponding disulfide and Cu(I). Cu(I) binds on the average only one sulfhydryl molecule, and a polymeric structure is suggested. A mixed valence state purple species absorbing at 520 nm and stable even in air is formed when approximately equivalent amounts of Cu(I), Cu(II), and d-penicillamine are present in neutral solutions. In the absence of oxygen and in the presence of 0.1 n base d-penicillamine forms a 2:1 complex with Cu(II) that is stable for hours. Absorption and circular dichroism are reported for the above species and the Cu(II) complexes of l-cystinediamide and l-cystinylbisglycine.     

Copper(I) transfer into apo-stellacyanin using copper(I)-thiourea as a copper-thionein model.

The direct incorporation of Cu(I) from [Cu(I)(thiourea)3]Cl, a structural analogue of Cu-thionein, into apo-stellacyanin, was successful both aerobically and anaerobically. A characteristic c.d. band of Cu(I)-stellacyanin at 270 nm (0 = -12.5 X 10(3) degrees X cm2 X dmol-1) was seen. On oxidation with hexacyanoferrate(III) or by air, the correct Cu(II) binding into the active centre of this 'Type 1' Cu-protein was deduced from chiroptical measurements which were supported by e.p.r. data. Thus Cu-thiourea turned out to be an excellent Cu(I)-donor in aqueous systems for the complete reconstitution of mononuclear Blue copper proteins.     

Aggregation of alpha-synuclein induced by the Cu,Zn-superoxide dismutase and hydrogen peroxide system

Alpha-synuclein is a major component of the abnormal protein aggregation in Lewy bodies of Parkinson's disease (PD) and senile plaques of Alzheimer's disease (AD). Previous studies have shown that the aggregation of alpha-synuclein was induced by copper (II) and H(2)O(2) system. Since copper ions could be released from oxidatively damaged Cu,Zn-superoxide dismutase (SOD), we investigated the role of Cu,Zn-SOD in the aggregation of alpha-synuclein. When alpha-synuclein was incubated with both Cu,Zn-SOD and H(2)O(2), alpha-synuclein was induced to be aggregated. This process was inhibited by radical scavengers and spin trapping agents such as 5,5'-dimethyl 1-pyrolline N-oxide and tert-butyl-alpha-phenylnitrone. Copper chelators, diethyldithiocarbamate and penicillamine, also inhibited the Cu,Zn-SOD/H(2)O(2) system-induced alpha-synuclein aggregation. These results suggest that the aggregation of alpha-synuclein is mediated by the Cu,Zn-SOD/H(2)O(2) system via the generation of hydroxyl radical by the free radical-generating function of the enzyme. The Cu,Zn-SOD/H(2)O(2)-induced alpha-synuclein aggregates displayed strong thioflavin-S reactivity, reminiscent of amyloid. These results suggest that the Cu,Zn-SOD/H(2)O(2) system might be related to abnormal aggregation of alpha-synuclein, which may be involved in the pathogenesis of PD and related disorders.     

Raman and IR study on copper binding of histamine

A comparative Raman and FTIR study of histamine (Hm), a small hormone present in a wide selection of living organisms, and its complexes with copper(II) at different pH values was carried out. Both the Raman and IR spectra present some marker bands useful for the identification of the structure of the species predominating in the Cu(II) aqueous and alcoholic systems. In particular, Raman spectroscopy appears to be a useful tool for analyzing the tautomeric equilibrium of the imidazole ring of Hm, because some bands (i.e., nuC(4)dbond;C(5)) appear at different wavenumbers, depending on whether the imidazole moiety is in the N(tau)-H (tautomer I) or N(pi)-H (tautomer II) protonated form. In aqueous solutions the manner in which Hm binds to Cu(II) depends on the pH. At basic pH the most relevant species formed are a dimer, [Cu(2)L(2)H(-2)](2+), and a monomeric complex, [CuL](2-) or [CuL(2)](+). On the contrary, by decreasing the pH, Hm acts as a mono- or bidentate ligand, giving rise to two types of monomeric complexes, [CuLH](2-) and [CuL](2-) or [CuL(2)](+). With respect to the Cu(II)-Hm alcoholic system, both the aminic group and the imidazole ring (tautomer I) take part in the Cu(II) coordination, leading to the formation of the [CuL](2-) or [CuL(2)](+) monomeric complex.     

Characterization of the binding interface between the copper chaperone Atx1 and the first cytosolic domain of Ccc2 ATPase

The interaction of the copper chaperone Atx1 and the first cytosolic domain of Ccc2 ATPase, Ccc2a, was investigated by NMR in solution. In particular, a solution of Cu(I)-15NAtx1 was titrated with apo-Ccc2a, and, vice versa, a solution of Cu(I)-15NCcc2a was titrated with apo-Atx1. By following the 15N and 1H chemical shifts, a new species is detected in both experiments. This species is the same in both titrations and is in fast exchange with the parent species on the NMR time scale. Nuclear relaxation data are consistent with the formation of an adduct. Judging from the nuclear Overhauser effect spectroscopy patterns, the structure of Cu(I)-15NCcc2a in the presence of apo-Atx1 is not significantly altered, whereas Cu(I)-15NAtx1 in the presence of apo-Ccc2a experiences some changes with respect to both the apoproteins and the Cu(I)-loaded proteins. The structure of the Cu(I)-15NAtx1 moiety in the adduct was obtained from 1137 nuclear Overhauser effects to a final root mean square deviation to the mean structure of 0.76 +/- 0.13 A for the backbone and 1.11 +/- 0.11 A for the heavy atoms. 15N and 1H chemical shifts suggest the regions of interaction that, together with independent information, allow a structural model of the adduct to be proposed. The apo form of Atx1 displays significant mobility in loops 1 and 5, the N-terminal part of helix alpha1, and the C-terminal part of helix alpha2 on the ms-micros time scale. These regions correspond to the metal binding site. Such mobility is largely reduced in the free Cu(I)-Atx1 and in the adduct with apo-Ccc2a. The analogous mobility of Ccc2a in both Cu(I) and apo forms is reduced with respect to Atx1. Such an adduct is relevant as a structural and kinetic model for copper transfer from Atx1 to Ccc2a in physiological conditions.     

Binding of copper(II) to carnosine: Raman and IR spectroscopic study

A comparative Raman and FTIR study of carnosine, a dipeptide present in several mammalian tissues, and its complexes with copper(II) at different pH values was carried out. The neutral imidazole ring gives rise to some bands that appear at different wavenumbers, depending on whether the imidazole ring is in the tautomeric form II or I. At pH 7 and 9 the molecule exists in equilibrium between the two tautomeric forms; tautomer I is predominant. Metal coordination is a factor that affects the tautomeric equilibrium, and the copper(II) coordination site can be monitored by using some Raman marker bands such as the vC(4)=C(5) band. On the basis of the vibrational results, conclusions can be drawn on the functional groups involved in the Cu(II) chelation and on the species existing in the Cu(II)-carnosine system. At neutral and basic pH the most relevant species formed when the Cu(II)/carnosine molar ratio is not very different from unity is a dimer, [Cu(2)L(2)H(-2)](0). In this complex the ligand coordinates the metal via the N (amino), O (carboxylate), and N (amide) donor atoms while the N(tau) nitrogen atoms of the imidazole rings (tautomer II) bridge the copper(II) ions. At a slightly acidic pH the two monomeric complexes [CuLH](2+) and [CuL](+) were present. In the former the imidazole ring takes part in the Cu(II) coordination in the tautomeric I form whereas in the latter it is protonated and not bound to Cu(II).     

Similarity and dissimilarity of thiols as anti-nitrosative agents in the nitric oxide-superoxide system

Concomitant production of nitric oxide and superoxide in biological systems has been proposed to generate numerous reactive oxygen and nitrogen species that cause oxidative and nitrosative stress. Thiols, especially glutathione, play an important role in cellular defense against radical species. In the present study, we investigated and compared the anti-nitrosative activity of a wide range of thiols in a simplified chemical system of co-generated nitric oxide and superoxide. Of the 13 thiols studied, three groups of thiols are distinguishable: (i) Group I includes cysteine and its four congeners (cysteine methyl ester, cysteine ethyl ester, homocysteine, cysteamine); they are subject to rapid oxidative decomposition and have the least anti-nitrosative activity. (ii) Group II consists of glutathione, penicillamine, tiopronin and mesna; they have the greatest effect on delaying the nitrosation reaction. (iii) Group III comprises N-acetylcysteine, N-acetylpenicillamine, captopril, and thioglycolate; they all have high pK_a for the mercapto group and show the strongest inhibitory effect on the rate and extent of nitrosation in the system studied.     

The hydrogen peroxide/copper ion system, but not other metal-catalyzed oxidation systems, produces protein-bound dityrosine

Dityrosine formation leads to the cross-linking of proteins intra- or intermolecularly. The formation of dityrosine in lens proteins oxidized by metal-catalyzed oxidation (MCO) systems was estimated by chemical and immunochemical methods. Among the four MCO systems examined (H(2)O(2)/Cu, H(2)O(2)/Fe-ethylenediaminetetraacetic acid (Fe-EDTA), ascorbate/Cu, ascorbate/Fe-EDTA), the treatment with H(2)O(2)/Cu preferentially caused dityrosine formation in the lens proteins. The success of oxidative protein modification with all the MCO systems was confirmed by carbonyl formation estimated using 2,4-dinitrophenylhydrazine. The loss of tyrosine by the MCO systems was partly due to the formation of protein-bound 3,4-dihydroxyphenylalanine. The formation of dityrosine specific to H(2)O(2)/Cu was confirmed by using poly-(Glu, Ala, Tyr) and N-acetyl-tyrosine as a substrate. The dissolved oxygen concentration in the H(2)O(2)/Cu system hardly affected the amount of dityrosine formation, suggesting that dityrosine generation by the H(2)O(2)/Cu system is independent of oxygen concentration. Moreover, the combination of copper ion with H(2)O(2) is the most effective system for dityrosine formation among various metal ions examined. The addition of reducing agents, glutathione or ascorbic acid, into the H(2)O(2)/Cu system suppressed the generation of the dityrosine moiety, suggesting effective quench of tyrosyl radicals by the reducing agents.     

Comparison of the catalytic oxidation of cysteine and o-dianisidine by cupric ion and ceruloplasmin

Several features of the catalytic oxidation of cysteine by ceruloplasmin and nonenzymic Cu(II) at pH 7 have been compared. The oxidation of cysteine by ceruloplasmin has several properties in common with the Cu(II) catalyzed oxidation of cysteine: pH maxima, thiol specificity, lack of inhibition by anions, and high sensitivity to inhibition by copper complexing reagents. These two catalysts differed in their molecular activity, in their ability to oxidize penicillamine and thioglycolate, and in that H₂O₂ was produced as a primary product only during Cu(II) oxidation. The oxidation of cysteine by ceruloplasmin was compared also with the ceruloplasmin catalyzed oxidation of o-dianisidine, a classical pH 5.5 substrate. The mechanism of the oxidation of cysteine by ceruloplasmin at pH 7 differed from that of o-dianisidine oxidation because the latter substrate was inhibited by anions but not by copper complexing agents. Spectral and other data suggest that during the ceruloplasmin reaction with cysteine there is a one electron transfer from cysteine to ceruloplasmin resulting in the specific reduction of type lb Cu(II).     

Synthesis and possible applications of biotin-linked copper clusters

The trifunctional aziridine XAMA-7 (CAS 57116-45-7) has been used to form crosslinks between a deep red-violet copper cluster of the type Cu(I)8Cu(II)6pen12Cl5- (pen=penicillamine) and molecules with biological activity such as d-biotin and proteins. A complex containing biotin, bovine serum albumin and the copper cluster displayed activity toward affinity columns of avidin on Agarose, and the red-violet pigment was immobilized on the gel. This interaction was completely blocked in gels which had been pretreated with d-biotin carboxylic acid. The free and biologically active versions of the cluster have some potential for biomedical applications. For example, the short-lived positron emitter 64Cu (suitable for positron tomography) may be carried in the cluster's structure. The cluster is paramagnetic, but it is a relatively weak effector of water proton spin-lattice relaxation. Other members of this structural group of inorganic compounds may have better magnetic properties, and the crosslinking reaction with aziridines appears to be generally applicable to the group.     

Nuclear magnetic resonance studies of the solution chemistry of metal complexes. 26. Mixed ligand complexes of cadmium, nitrilotriacetic acid, glutathione, and related ligands

The complexation of glutathione and related ligands by the nitrilotriacetic acid complex of Cd2+ (Cd(NTA)-) has been investigated by 1H NMR as a model for the coordination chemistry of Cd2+ and GSH in biological systems. Related ligands included glycine, glutamic acid, cysteine, N-acetylcysteine, penicillamine, N-acetylpenicillamine, mercaptosuccinic acid, and the S-methyl derivative of glutathione. The nature of the complexes formed was deduced from 1H NMR spectra of Cd(NTA)- and the ligands. Mixed ligand complexes (Cd(NTA)L) and single ligand complexes (CdLx) are formed with the thiol ligands, whereas only mixed ligand complexes form with glycine, glutamic acid and S-methylglutathione. Formation constants of the mixed and the single ligand complexes were determined from NMR data. The results indicate that formation constants for binding of a thiolate donor group by Cd2+, either as the free ion or in a coordinately unsaturated complex, are in the range 10(5)-10(6).     

The copper-enzyme dopamine beta-monooxygenase: studies on the ability of several metals to inhibit the enzyme activity and to replace the copper

The ability of several metals to inhibit dopamine beta-monooxygenase was measured and compared with their ability to compete with the binding of 64Cu to the water-soluble form of the bovine adrenal enzyme at pH 6.0. In the presence of an optimal concentration of copper (0.5 microM in the present assay system), an inhibition was observed upon addition of Hg(II), Zn(II), or Ni(II). Only a small fraction of the inhibition with these metals may be due to uncoupling of electron transport from hydroxylation. Preincubation of these metals with the Cu-depleted apoenzyme before addition of copper, revealed a stronger inhibition than if copper was added before the other metals. Hg(II), Zn(II), and Ni(II) also compete with the binding of 64Cu(II) to the protein. Hg(II) was the most effective and Ni(II) the least effective of these metals, both with respect to inhibition of the enzyme activity and to prevent the binding of 64Cu(II). Competition experiments on the binding of Zn(II) and 64Cu in the presence and absence of ascorbate, indicated i) a similar affinity of Cu(I) and Cu(II) to the native enzyme, and ii) a more rapid binding of Cu(I) than Cu(II) to the Cu-depleted and Zn-containing enzyme. Al(III), Fe(II), Mg(II), Mn(II), Co(II), Cd(II), and Pb(II) neither inhibited the enzyme activity nor competed with the binding of 64Cu(II) to the protein (Fe(II) was not tested for binding). Of those metals cited above only Cu(II)/Cu(I) was able to reactivate the apoenzyme.     

Role of the coordinating histidine in altering the mixed valency of Cu(A): an electron nuclear double resonance-electron paramagnetic resonance investigation

The binuclear Cu(A) site engineered into Pseudomonas aeruginosa azurin has provided a Cu(A)-azurin with a well-defined crystal structure and a CuSSCu core having two equatorial histidine ligands, His120 and His46. The mutations His120Asn and His120Gly were made at the equatorial His120 ligand to understand the histidine-related modulation to Cu(A), notably to the valence delocalization over the CuSSCu core. For these His120 mutants Q-band electron nuclear double resonance (ENDOR) and multifrequency electron paramagnetic resonance (EPR) (X, C, and S-band), all carried out under comparable cryogenic conditions, have provided markedly different electronic measures of the mutation-induced change. Q-band ENDOR of cysteine C(beta) protons, of weakly dipolar-coupled protons, and of the remaining His46 nitrogen ligand provided hyperfine couplings that were like those of other binuclear mixed-valence Cu(A) systems and were essentially unperturbed by the mutation at His120. The ENDOR findings imply that the Cu(A) core electronic structure remains unchanged by the His120 mutation. On the other hand, multifrequency EPR indicated that the H120N and H120G mutations had changed the EPR hyperfine signature from a 7-line to a 4-line pattern, consistent with trapped-valence, Type 1 mononuclear copper. The multifrequency EPR data imply that the electron spin had become localized on one copper by the His120 mutation. To reconcile the EPR and ENDOR findings for the His120 mutants requires that either: if valence localization to one copper has occurred, the spin density on the cysteine sulfurs and the remaining histidine (His46) must remain as it was for a delocalized binuclear Cu(A) center, or if valence delocalization persists, the hyperfine coupling for one copper must markedly diminish while the overall spin distribution on the CuSSCu core is preserved.     

X-ray absorption and NMR spectroscopic studies of CopZ,a copper chaperone in Bacillus subtilis: the coordination properties of the copper ion

XAS studies have been performed, under various experimental conditions, on a copper(I)-transporting protein, CopZ, of Bacillus subtilis. The copper(I) ion, reduced with dithiothreitol, is three-coordinate with three sulfur donor atoms, two of which presumably provided by the protein and one by dithiothreitol. If a molar excess of acetate (15 mM; 5:1 respect to CopZ) or citrate (6 mM; 2:1 respect to CopZ) is present in solution, the EXAFS spectra suggest the presence of a dimeric form involving a close contact between Cu(I) ions from two molecules, where Cu is still three-coordinate. (1)H and (15)N NMR data provide further structural details. If copper reduction is accomplished with ascorbate, the data indicate that one oxygen of ascorbate enters in the first-coordination sphere of copper, together with two sulfur atoms, in a dimeric form of the protein. These results are instructive and have been discussed with respect to the molecular basis of copper trafficking.