Unification of the copper(I) binding affinities of the metallo-chaperones Atx1, Atox1, and related proteins: detection probes and affinity standards

Literature estimates of metal-protein affinities are widely scattered for many systems, as highlighted by the class of metallo-chaperone proteins, which includes human Atox1. The discrepancies may be attributed to unreliable detection probes and/or inconsistent affinity standards. In this study, application of the four Cu(I) ligand probes bicinchoninate, bathocuproine disulfonate, dithiothreitol (Dtt), and glutathione (GSH) is reviewed, and their Cu(I) affinities are re-estimated and unified. Excess bicinchoninate or bathocuproine disulfonate reacts with Cu(I) to yield distinct 1:2 chromatophoric complexes [Cu(I)L(2)](3-) with formation constants β(2) = 10(17.2) and 10(19.8) m(-2), respectively. These constants do not depend on proton concentration for pH ≥7.0. Consequently, they are a pair of complementary and stable probes capable of detecting free Cu(+) concentrations from 10(-12) to 10(-19) m. Dtt binds Cu(I) with K(D) ～10(-15) m at pH 7, but it is air-sensitive, and its Cu(I) affinity varies with pH. The Cu(I) binding properties of Atox1 and related proteins (including the fifth and sixth domains at the N terminus of the Wilson protein ATP7B) were assessed with these probes. The results demonstrate the following: (i) their use permits the stoichiometry of high affinity Cu(I) binding and the individual quantitative affinities (K(D) values) to be determined reliably via noncompetitive and competitive reactions, respectively; (ii) the scattered literature values are unified by using reliable probes on a unified scale; and (iii) Atox1-type proteins bind Cu(I) with sub-femtomolar affinities, consistent with tight control of labile Cu(+) concentrations in living cells.     

Fibrillation of hen egg white lysozyme triggers reduction of copper(II)

Copper is known to exert diverse effects on the self-association of proteins and has been found in amyloid deposits that are involved in neurodegenerative disease processes. The effects of the metal ion on the protein during fibrillation were investigated by fluorescence, circular dichroism spectroscopy and fluorescence microscopy. We report for the first time, the complete reduction of Cu(II)→Cu(I) in vitro during fibrillation of hen egg white lysozyme at pH 7. This was confirmed by the lack of any signal for Cu(II) in electron paramagnetic resonance spectroscopy and quantification of Cu(I) was achieved by a bathocuproine disulfonate based assay.     

A new look at a time-worn system: oxidation of CuZn-SOD by H2O2

This work summarizes observations from numerous investigators on the reaction of the copper-zinc superoxide dismutase with hydrogen peroxide at physiological pH in order to propose a likely sequence of events that leads to 2-oxo-histidine formation, copper loss, inactivation, and random and site-specific peptide fragmentation. New data is presented for the bovine liver enzyme that indicate copper is lost as the copper(I) form which immediately reacts with bathocuproine disulfonate to form the characteristic complex that absorbs at 485 nm. Studies in TRIS buffer ruled out the loss of copper(II) followed by reduction of the high potential copper(II)-bathocuproine disulfonate complex by buffer because TRIS is known not to reduce this complex. The rate of loss of copper(I) is not affected by the spin trap, 5,5'-dimethylpyrolline-N-oxide (DMPO), nor by replacing oxygen with argon in the reaction. In addition, changes in the native electrophoretic pattern that are correlated with copper loss and not peptide fragmentation are also unaffected by DMPO, argon, EDTA, or DTPA. These data are taken as indirect evidence that the formation of 2-oxo-histidine is the first oxidative event, unaffected by DMPO, that occurs at the bound oxidant and leads to loss of copper(I). Peptide fragmentation and the peroxidative activity of the dismutase are discussed in light of these observations.     

Inactivation of dopamine β-monooxygenase by hydrogen peroxide and by ascorbate

The inactivation of the water-soluble form of bovine adrenal dopamine β-monooxygenase by H₂O₂ and by ascorbate was studied. Inactivation by H₂O₂ was slow for the copper-free apoenzyme, but addition of copper gave a rapid inactivation. The results presented indicate that the enzyme-bound copper during this inactivation catalyzes partial destruction of its own binding site. The reaction orders for the inactivation by H₂O₂ seem to be 1.0 with respect to the enzyme and in the range 0.6 to 0.8 with respect to H₂O₂. The rate of inactivation obtained in the presence of ascorbate increases with addition of copper and is faster than that obtained by similar concentrations of H₂O₂. The data could not, however, be used to decide whether the inactivation by ascorbate was catalyzed by the enzymebound copper. The inactivation reaction in the presence of ascorbate seems to be of first order with respect to ascorbate at ascorbate concentrations less than 40 μm and decreases toward zero as the ascorbate concentration is increased. Experiments with the Cu(I)-chelator, bathocuproine disulfonate, revealed that inactivation led to weaker binding of copper to the protein, and this effect was more pronounced with H₂O₂ than with ascorbate.     

High Cu(I) and low proton affinities of the CXXC motif of Bacillus subtilis CopZ

CopZ, an Atx1-like copper chaperone from the bacterium Bacillus subtilis, functions as part of a complex cellular machinery for Cu(I) trafficking and detoxification, in which it interacts specifically with the transmembrane Cu(I)-transporter CopA. Here we demonstrate that the cysteine residues of the MXCXXC Cu(I)-binding motif of CopZ have low proton affinities, with both exhibiting pK(a) values of 6 or below. Chelator competition experiments demonstrated that the protein binds Cu(I) with extremely high affinity, with a small but significant pH-dependence over the range pH 6.5-8.0. From these data, a pH-corrected formation constant, beta(2)= approximately 6 x 10(22) M(-2), was determined. Rapid exchange of Cu(I) between CopZ and the Cu(I)-chelator BCS (bathocuproine disulfonate) indicated that the mechanism of exchange does not involve simple dissociation of Cu(I) from CopZ (or BCS), but instead proceeds via the formation of a transient Cu(I)-mediated protein-chelator complex. Such a mechanism has similarities to the Cu(I)-exchange pathway that occurs between components of copper-trafficking pathways.     

Isolation of mannose-binding proteins from human and rat liver

The interaction of e-aq., CO2-. and one-electron reduced nitroaromatics (RNO2-.) with ascorbate oxidase (AAO) was studied in aqueous solution at pH 6.0 and 7.5 by using the technique of pulse radiolysis. From observations at 330, 410 and 610 nm, interaction of e-aq. and CO2-. with AAO results in non-specific reduction of the protein followed by reduction of Type 1 Cu in a rate-determining intramolecular step. Only a few per cent of the reducing equivalents ultimately results in reduction of Type 1 Cu. With large excesses of reducing equivalents (e-aq. and CO2-.) with respect to the copper concentration, the amount of Type 1 copper reduced never exceeds 50% of the total amount of Type 1 copper after a single radiation pulse. With less-powerful reducing agents, e.g. RNO2-. reduction of Type 1 Cu occurs via a bimolecular step, and there is no evidence for formation of radicals on protein residues. From observations at 330 nm it is evident that Type 2 and/or Type 3 Cu may also be reduced along with Type 1 Cu. Almost stoichiometric reduction of AAO by RNO2-. was observed, e.g. the protein accepts 6-7 reducing equivalents. It is inferred that the various types of redox couples Cu2+/Cu+ are in equilibrium and that intramolecular electron transfer between the different types of Cu is not rate-determining when using RNO2-. as reducing agent.     

Modulation of copper accumulation and copper-induced toxicity by antioxidants and copper chelators in cultured primary brain astrocytes

Copper is essential for several important cellular processes, but an excess of copper can also lead to oxidative damage. In brain, astrocytes are considered to play a pivotal role in the copper homeostasis and antioxidative defence. To investigate whether antioxidants and copper chelators can modulate the uptake and the toxicity of copper ions in brain astrocytes, we used primary astrocytes as cell culture model. These cells accumulated substantial amounts of copper during exposure to copper chloride. Copper accumulation was accompanied by a time- and concentration-dependent loss in cell viability, as demonstrated by a lowering in cellular MTT reduction capacity and by an increase in membrane permeability for propidium iodide. During incubations in the presence of the antioxidants ascorbate, trolox or ebselen, the specific cellular copper content and the toxicity in copper chloride-treated astrocyte cultures were strongly increased. In contrast, the presence of the copper chelators bathocuproine disulfonate or tetrathiomolybdate lowered the cellular copper accumulation and the copper-induced as well as the ascorbate-accelerated copper toxicity was fully prevented. These data suggest that predominantly the cellular content of copper determines copper-induced toxicity in brain astrocytes.     

Ascorbate-induced high-affinity binding of copper to cytosolic proteins

Copper chaperones are necessary for intracellular trafficking of copper to target proteins. This is probably because the milieu inside the cell has a large capacity for sequestering this metal. By fluorometry using a fluorescent Cu(II) chelator and by centrifugal ultrafiltration, we have studied copper binding of the whole cytosolic proteins from mouse brain and liver, and found that their binding capacity and affinity for copper were markedly increased by ascorbate. Brain cytosolic protein bound, with high affinity, 63 nmol of copper/mg, more than half of which was redox-inactive, as indicated by its inability to catalyze oxidation of ascorbate. Most of the bound copper was in the Cu(I) state, coordinating to thiol groups of protein. Cytosolic protein competed for copper more strongly than GSH when compared at their relative concentrations in tissues. The results taken together suggest that protein thiols of cytosol can strongly sequester copper.     

The enzyme-bound copper of dopamine beta-monooxygenase. Reaction with copper chelators, preparation of the apoprotein, and kinetics of the reconstitution by added copper.

The enzyme-bound copper of dopamine beta-monooxygenase reacted rapidly with the chelator bathocuproine disulfonate; the reaction in the presence of ascorbate was completed in 2 min at 25 degrees C with 1mM chelator. This reaction and also the reaction with EDTA could be used to prepare the apoenzyme, which in both cases was completely reactivated in less than 10 s. The reactivation data gave apparent Michaelis constants for copper 0.03 -- 0.2 micron. Trace amounts of copper in buffers and assay mixtures gave significant reactivation without added copper, unless they had been treated with a chelating resin. Titrations using the different chelation rates of free and enzyme-bound copper indicated that four copper atoms are bound per enzyme molecule of four subunits. The native enzyme was more stable against thermal inactivation than the apoenzyme, but this stability was only partially restored by addition of copper to the apoenzyme.     

Effect of chelating agents and superoxide on human neutrophil NAD(P)H oxidation

NAD(P)H oxidation is frequently measured to assay the activity of the neutrophil O-2-generating oxidase. It was found that 10(-4) M ethylene glycol bis (beta-aminoethyl ether)-N-N'-tetraacetic acid (EGTA) increased NAD(P)H oxidation by the 27,000 g granule fraction of resting and stimulated human neutrophils without altering net O-2 production. The commonly used chelating agents EDTA and diethylene triamine pentaacetic acid had similar effects. The addition of superoxide dismutase eliminated the effect of the chelating agents and thus demonstrated that the stimulated reaction was dependent upon O-2. KCN and bathophenanthroline disulfonate, an iron-chelating agent, prevented O-2-dependent NADPH oxidation by neutrophil granule fractions in the presence of EGTA. In contrast, bathocuproine disulfonate, a copper-chelating agent, mimicked the EGTA effect. The effects of both bathophenanthroline disulfonate and bathocuproine disulfonate were completely abolished when the agents were saturated with iron and copper, respectively. All the chelating agents studied, except bathophenonthroline disulfonate, also promoted O-2-dependent NADPH oxidation in a system wherein O-2 was generated by xanthine oxidase. Thus, commonly used chelating agents, by interacting with available iron and copper, may alter the apparent stoichiometry of the neutrophil O-2-generating oxidase and artifactually increase NADPH oxidation in other systems where O-2 is present.     

Golgi membranes from liver express an ATPase with femtomolar copper affinity, inhibited by cAMP-dependent protein kinase

Copper-stimulated P-type ATPases are essential in the fine-tuning of intracellular copper. In the present work we characterized a copper-dependent ATPase hydrolysis in a native Golgi-enriched preparation from liver and investigated its modulation by cyclic AMP-dependent protein kinase (PKA). The very high-affinity Atp7b copper pump presented here shows a K(0.5) for free copper of 2.5×10(-17) M in bathocuproine disulfonate/copper buffer and ATP hydrolysis was inhibited 50% upon stimulation of PKA pathway, using forskolin, cAMP or cholera toxin. Incubation with PKA inhibitor (PKAi(5-24) peptide) raises Cu(I)-ATPase activity by 50%. Addition of purified PKA α-catalytic subunit increases K(0.5) for free copper (6.2×10(-17) M) without modification in the affinity for ATP in the low-affinity range of the substrate curve (～1 mM). The Hill coefficient for free copper activation also remains unchanged if exogenous PKA is added (2.7 and 2.3 in the absence and presence of PKA, respectively). The results demonstrate that this high-affinity copper pump in its natural environment is a target of the liver PKA pathway, being regulatory phosphorylation able to influence both turnover rate and ion affinity.     

Copper modifies liver microsomal UDP-glucuronyltransferase activity through different and opposite mechanisms

Treatment of hepatic microsomes with Fe(3+)/ascorbate activates UDP-glucuronyltransferase (UGT), a phenomenon totally prevented and reversed by reducing agents. At microM concentrations, iron and copper ions catalyze the formation of ROS through Fenton and/or Haber-Weiss reactions. Unlike iron ions, indiscriminate binding of copper ions to thiol groups of proteins different from the specialized copper-binding proteins may occur. Thus, we hypothesize that incubation of hepatic microsomes with the Cu(2+)/ascorbate system will lead to both UGT oxidative activation and Cu(2+)-binding induced inhibition, simultaneously. We studied the effects of Cu(2+) alone and in the presence of ascorbate on rat liver microsomal UGT activity. Our results show that the effects of both copper alone and in the presence of ascorbate were copper ion concentration- and incubation time-dependent. At very low Cu(2+) (25nM), this ion did not modify UGT activity. In the presence of ascorbate, however, UGT activity was increased. At higher copper concentrations (10 and 50microM), this ion led to UGT activity inhibition. In the presence of ascorbate, 10microM Cu(2+) activated UGT at short incubation periods but inhibited this enzyme at longer incubation times; 50microM Cu(2+) only inhibited UGT activity. Thiol reducing agent 2,4-dithiothreitol prevented and reversed UGT activation while EDTA prevented both, UGT activation and inhibition. Our results are consistent with a model in which Cu(2+)-induced oxidation of UGT leads to the activation of the enzyme, while Cu(2+)-binding leads to its inhibition. We discuss physiological and pathological implications of these findings.     

Cu(I)- and proton-binding properties of the first N-terminal soluble domain of Bacillus subtilis CopA

CopA, a P-type ATPase transporter involved in copper detoxification in Bacillus subtilis, contains two soluble Atx1-like domains separated by a short linker at its N-terminus, an arrangement that occurs widely in copper transporters from both prokaryotes and eukaryotes. Both domains were previously found to bind Cu(I) with very high affinity. Above a level of 1 Cu(I) per CopAab, dimerization occurred, leading to a highly luminescent multinuclear Cu(I) species [Singleton C & Le Brun NE (2009) Dalton Trans, 688-696]. To try to understand the contributions of each domain to the complex Cu(I)-binding behaviour of this and related proteins, we purified a wild-type form of the first domain (CopAa). In isolation, the domain bound Cu(I) with very high affinity (K = ～ 1 × 10(18) m(-1) ) and underwent Cu(I)-mediated protein association, resulting in a mixture of dimer and tetramer species. Addition of further Cu(I) up to 1 Cu(I) per CopAa monomer led to a weakly luminescent species, whereas further additions [2 Cu(I) per CopAa monomer] resulted in protein unfolding. Analysis of the MTCAAC binding motif Cys residue acid-base properties revealed pK(a) values of 5.7 and 7.3, consistent with the pH dependence of Cu(I) binding, and with the proposal that low proton affinity is associated with high Cu(I) affinity. Finally, Cu(I) exchange between CopAa and the chelator bathocuproine sulfonate revealed rapid exchange in both directions, demonstrating an interaction between the protein and the chelator that catalyses metal ion transfer. Overall, CopAa exhibits similarities to CopAab in terms of affinity and complexity of Cu(I) binding, but the details of Cu(I) binding are distinct.     

Pulse-radiolysis studies on the interaction of one-electron reduced species with blue oxidases. Reduction of type-2-copper-depleted ascorbate oxidase.

The interaction of one-electron reduced metronidazole (ArNO2.-) with native and Type-2-copper-depleted ascorbate oxidase were studied in buffered aqueous solution at pH 6.0 and 7.4 by using the technique of pulse radiolysis. With ArNO2.-, reduction of Type 1 copper of the native enzyme and of the Type-2-copper-depleted ascorbate oxidase occurs via a bimolecular step and at the same rate. Whereas the native protein accepts, in the absence of O2, 6-7 reducing equivalents, Type-2-copper-depleted ascorbate oxidase accepts only 3 reducing equivalents with stoichiometric reduction of Type 1 copper. On reaction of O2.- with ascorbate oxidase under conditions of [O2.-] much greater than [ascorbate oxidase], removal of Type 2 copper results in reduction of all the Type 1 copper atoms, in contrast with reduction of the equivalent of only one Type 1 copper atom in the holoprotein. From observations at 610 nm, the rate of reduction of ascorbate oxidase by O2.- is not dependent on the presence of Type 2 copper. For the holoprotein, no significant optical-absorption changes were observed at 330 nm. It is proposed that electrons enter the protein via Type 1 copper in a rate-determining step followed by a fast intramolecular transfer of electrons within the protein. For the Type-2-copper-depleted protein, intramolecular transfer within the protein, however, is slow or does not occur. In the presence of O2, it is also suggested that re-oxidation of the partially reduced holoprotein occurs at steady state, as inferred from the observations at 330 nm and 610 nm. The role of Type 2 copper in ascorbate oxidase is discussed in terms of its involvement in redistribution of electrons within the protein or structural considerations.     

The dose-dependent effect of copper-chelating agents on the kinetics of peroxidation of low-density lipoprotein (LDL)

Copper-induced peroxidation of lipoproteins involves continuous production of free radicals via a redox cycle of copper. Formation of Cu(I) during Cu(II)-induced peroxidation of LDL was previously demonstrated by accumulation of the colored complexes of Cu(I) in the presence of one of the Cu(I)-specific chelators bathocuproine (BC) or neocuproine (NC). All the studies conducted thus far employed high concentrations of these chelators (chelator/Cu(II) > 10). Under these conditions, at low copper concentrations the chelators prolonged the lag preceding oxidation, whereas at high copper concentrations the chelators shortened the lag. In an attempt to gain understanding of these non-monotonic effects, we have studied systematically the peroxidation of LDL (0.1 microM, 50 microg protein/mL) at varying concentrations of NC or BC over a wide range of concentrations of the chelators and copper. These studies revealed that: (i) At copper concentrations of 5 microM and below, NC prolonged the lag in a monotonic, dose-dependent fashion typical for other complexing agents. However, unlike with other chelators, the maximal rate of oxidation was only slightly reduced (if at all). (ii) At copper concentrations of 15 microM and above, the addition of about 20 microM NC or BC resulted in prolongation of the lag, but this effect became smaller at higher concentrations of the chelators, and at yet higher concentrations the lag became much shorter than that observed in the absence of chelators. Throughout the whole range of NC concentrations, the maximal rate of peroxidation increased monotonically upon increasing the NC concentration. (iii) Unlike in the absence of chelators, the prooxidative effect of copper did not exhibit saturation with respect to copper, up to copper concentrations of 30 microM. Based on these results we conclude that the copper-chelates can partition into the hydrophobic core of LDL particles and induce peroxidation by forming free radicals within the core. This may be significant with respect to the understanding of the possible mechanisms of peroxidation by chelated transition metals in vivo.     

Copper Proteins and Oxygen : Correlations between structure and function of the copper oxidases

A comprehensive survey of the interaction of the copper proteins and oxygen is presented including a correlation of structure, function, and other properties of the known copper oxidases and of hemocyanin. The origin of their blue color and the structure of copper complexes and copper proteins are related to the oxidation state of copper ion and relevant electronic transitions probably arising from the formation of charge transfer complexes. The oxygen reactions of hemocyanin, ceruloplasmin, and cytochrome oxidase show half-saturation values far below the other Cu enzymes. The formation of hydrogen peroxide as a reaction product is associated with the presence of one Cu atom per oxidase molecule or catalytic system. Water is the corresponding product of the other Cu oxidases with four or more Cu atoms per molecule, except for monoamine oxidase. Mechanisms for the oxidase action of the two and four electron transfer Cu oxidases and tyrosinase are proposed. These reactions account for the number, the oxidation-reduction potential, and the oxidation state of Cu in the resting enzyme, the cyclical change from Cu(II) to Cu(I), the diatomic nature of O₂, the sequence of the oxidation and reduction reactions, and other salient features. The catalytic reactions involved in the oxidation of ascorbic acid by plant ascorbate oxidase, ceruloplasmin, and Cu(II) are compared. Finally the substrate specificity, inhibitory control, and the detailed mechanism of the oxidase activity of ceruloplasmin are summarized.     

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.     

Interaction of ascorbate oxidase with inorganic anions

From the peelings of cucumber Cucumis sativus and marrow squash Cucurbita pepo var. giramontia highly purified ascorbate oxidase preparations were obtained. Molecular weights, optical and EPR spectra, total copper contents and different type copper contents of the both proteins were similar. The effects of NaN3, KCN, I- and F- on the optical and EPR spectra of the proteins were studied. The incubation of ascorbate oxidase with these anions lead to the partial reduction of the copper. The data obtained indicate that F- is bound to the copper atoms of the type 2, and that N5- modifies surroundings of these copper atoms. The copper atoms of types 1 and 2 in both ascorbate oxidases, unlike fungal laccase, are completely reduced under effect of CN-. The bleaching of ascorbate oxidase, observed in alkaline media involves also increasing of the intensity of the band at 330 nm. The results show that three types of copper in ascorbate oxidase have various sensitivities to the inorganic anions. These data are compared with results observed for another blue copper-containing enzymes, such as laccases and ceruloplasmin.     

Redox properties and acid-base equilibria of zucchini mavicyanin

The reduction potential of mavicyanin isolated from zucchini peelings, which is a blue copper protein belonging to the subclass of the phytocyanins, has been determined through direct electrochemistry as a function of temperature and pH. The enthalpy and entropy changes accompanying protein reduction were found to be very similar with those determined previously for other phytocyanins and to differ remarkably from those of azurins and plastocyanins. This finding contributes to further characterize phytocyanins as a distinct cupredoxins family also on thermodynamic grounds and improves our understanding of how the reduction potential of these metal centers in proteins is modulated by coordinative and solvation properties. The E degrees' of mavicyanin is found to be sensitive to two acid-base equilibria at the extremes of pH. One occurs below pH 4, and is related to the protonation and detachment from the Cu(I) center of a histidine ligand. The other, observed above pH 8, causes a remarkable change in the electrostatic potential and/or the field strength around the copper.