Quenching of bathocuproine disulfonate fluorescence by Cu(I) as a basis for copper quantification

In this paper we report the up to now ignored fluorescence properties of the specific Cu(I)-chelator bathocuproine disulfonate and their application in assays of total copper and Cu(I). The method is based on the linear quenching of the bathocuproine disulfonate emission at 770 nm (lambda(ex)580 nm) by increasing concentrations of Cu(I), at pH 7.5. Copper concentrations as low as 0.1 microM can be determined. Other metal ions (iron, manganese, zinc, cadmium, cobalt, nickel) do not interfere. The procedure for total copper determination in proteins includes HCl treatment to release the copper, neutralization to pH 7.5 in the presence of citrate to stabilize the copper, and reduction of the copper to Cu(I) by ascorbate in the presence of the chelator. This assay gave results coincident with the analysis by atomic absorption spectroscopy in two selected proteins. In addition, conditions are described (omitting HCl treatment and reduction by ascorbate) for direct measurement of Cu(I) in native proteins, as illustrated for the Escherichia coli NADH dehydrogenase-2. Data show that the fluorometric assays described in this paper are simple and convenient procedures for total copper and direct Cu(I) quantification in determined biological samples.     

Binding of copper(I) by the Wilson disease protein and its copper chaperone

The Wilson disease protein (WND) is a transport ATPase involved in copper delivery to the secretory pathway. Mutations in WND and its homolog, the Menkes protein, lead to genetic disorders of copper metabolism. The WND and Menkes proteins are distinguished from other P-type ATPases by the presence of six soluble N-terminal metal-binding domains containing a conserved CXXC metal-binding motif. The exact roles of these domains are not well established, but possible functions include exchanging copper with the metallochaperone Atox1 and mediating copper-responsive cellular relocalization. Although all six domains can bind copper, genetic and biochemical studies indicate that the domains are not functionally equivalent. One way the domains could be tuned to perform different functions is by having different affinities for Cu(I). We have used isothermal titration calorimetry to measure the association constant (K(a)) and stoichiometry (n) values of Cu(I) binding to the WND metal-binding domains and to their metallochaperone Atox1. The association constants for both the chaperone and target domains are approximately 10(5) to 10(6) m(-1), suggesting that the handling of copper by Atox1 and copper transfer between Atox1 and WND are under kinetic rather than thermodynamic control. Although some differences in both n and K(a) values are observed for variant proteins containing less than the full complement of six metal-binding domains, the data for domains 1-6 were best fitted with a single site model. Thus, the individual functions of the six WND metal-binding domains are not conferred by different Cu(I) affinities but instead by fold and electrostatic surface properties.     

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.     

Cu(I) binding and transfer by the N terminus of the Wilson disease protein

Wilson and Menkes diseases are genetic disorders of copper metabolism caused by mutations in the Wilson (WND) and Menkes (MNK) copper-transporting P1B-type ATPases. The N termini of these ATPases consist of six metal binding domains (MBDs). The MBDs interact with the copper chaperone Atox1 and are believed to play roles in catalysis and in copper-mediated cellular relocalization of WND and MNK. Although all six MBDs have similar folds and bind one Cu(I) ion via a conserved CXXC motif, biochemical and genetic data suggest that they have distinct functions. Most studies aimed at characterizing the MBDs have employed smaller polypeptides consisting of one or two domains. The role of each MBD is probably defined by its environment within the six-domain N terminus, however. To study the properties of the individual domains within the context of the intact Wilson N terminus (N-WND), a series of variants in which five of the six metal binding CXXC motifs are mutated to SXXS was generated. For each variant, the Cu(I) binding affinity and the ability to exchange Cu(I) with Atox1 were investigated. The results indicate that Atox1 can deliver Cu(I) to and remove Cu(I) from each MBD, that each MBD has stronger Cu(I) retention properties than Atox1, and that all of the MBDs as well as Atox1 have similar K(Cu) values of (2.2-6.3) x 10(10) m(-1). Therefore, the specific role of each MBD is not conferred by its position within the intact N-WND but may be related to interactions with other domains and partner proteins.     

Kinetic analysis of the interaction of the copper chaperone Atox1 with the metal binding sites of the Menkes protein

Excess copper is effluxed from mammalian cells by the Menkes or Wilson P-type ATPases (MNK and WND, respectively). MNK and WND have six metal binding sites (MBSs) containing a CXXC motif within their N-terminal cytoplasmic region. Evidence suggests that copper is delivered to the ATPases by Atox1, one of three cytoplasmic copper chaperones. Attempts to monitor a direct Atox1-MNK interaction and to determine kinetic parameters have not been successful. Here we investigated interactions of Atox1 with wild-type and mutated pairs of the MBSs of MNK using two different methods: yeast two-hybrid analysis and real-time surface plasmon resonance (SPR). A copper-dependent interaction of Atox1 with the MBSs of MNK was observed by both approaches. Cys to Ser mutations of conserved CXXC motifs affected the binding of Atox1 underlining the essentiality of Cys residues for the copper-induced interaction. Although the yeast two-hybrid assay failed to show an interaction of Atox1 with MBS5/6, SPR analysis clearly demonstrated a copper-dependent binding with all six MBSs highlighting the power and sensitivity of SPR as compared with other, more indirect methods like the yeast two-hybrid system. Binding constants for copper-dependent chaperone-MBS interactions were determined to be 10-5-10-6 m for all the MBSs representing relatively low affinity binding events. The interaction of Atox1 with pairs of the MBSs was non-cooperative. Therefore, a functional difference of the MBSs in the MNK N terminus cannot be attributed to cooperativity effects or varying affinities of the copper chaperone Atox1 with the MBSs.     

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.     

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.     

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.     

Protein disulfide isomerase, a multifunctional protein chaperone, shows copper-binding activity

Protein disulfide isomerase (PDI) is a 55 kDa multifunctional protein of the endoplasmic reticulum (ER) involved in protein folding and isomerization. In addition to the chaperone and catalytic functions, PDI is a major calcium-binding protein of the ER. Although the active site of PDI has a similar motif CXXC to the Cu-binding motif in Wilson and Menkes proteins and in other copper chaperones, there has been no report on any metal-binding capability of PDI other than calcium binding. We present evidence that PDI is a copper-binding protein. In the absence of reducing agent freshly reduced PDI can bind a maximum of 4 mol of Cu(II) and convert to Cu(I). These bound Cu(I) are surface exposed as they can be competed readily by BCS reagent, a Cu(I) specific chelator. However, when the binding is performed using the mixture of Cu(II) and 1mM DTT, the total number of Cu(I) bound increases to 10 mol/mol, and it is slower to react with BCS, indicating a more protected environment. In both cases, the copper-bound forms of PDI exist as tetramers while apo-protein is a monomer. These findings suggest that PDI plays a role in intracellular copper disposition.     

Interplay between glutathione,Atx1 and copper. 1. Copper(I) glutathionate induced dimerization of Atx1

Copper is both an essential element as a catalytic cofactor and a toxic element because of its redox properties. Once in the cell, Cu(I) binds to glutathione (GSH) and various thiol-rich proteins that sequester and/or exchange copper with other intracellular components. Among them, the Cu(I) chaperone Atx1 is known to deliver Cu(I) to Ccc2, the Golgi Cu–ATPase, in yeast. However, the mechanism for Cu(I) incorporation into Atx1 has not yet been unraveled. We investigated here a possible role of GSH in Cu(I) binding to Atx1. Yeast Atx1 was expressed in Escherichia coli and purified to study its ability to bind Cu(I). We found that with an excess of GSH [at least two GSH/Cu(I)], Atx1 formed a Cu(I)-bridged dimer of high affinity for Cu(I), containing two Cu(I) and two GSH, whereas no dimer was observed in the absence of GSH. The stability constants (log β) of the Cu(I) complexes measured at pH 6 were 15–16 and 49–50 for CuAtx1 and Cu₂^I(GS⁻)₂(Atx1)₂, respectively. Hence, these results suggest that in vivo the high GSH concentration favors Atx1 dimerization and that Cu₂^I(GS⁻)₂(Atx1)₂ is the major conformation of Atx1 in the cytosol.     

Homocysteine promotes the LDL oxidase activity of ceruloplasmin

Ceruloplasmin (CP) oxidises low density lipoprotein (LDL). The oxidising potential depends on the formation of Cu(+)-CP which is redox-cycled during oxidation. Homocysteine (HCY) reduces free Cu(2+), potentiating its cell-damaging property. We show that HCY enhanced LDL oxidation by CP, but did not activate the LDL oxidising potential of Cu(2+)-diamine oxidase. Selective removal of the redox-active Cu(2+) abolished the LDL oxidase activity of CP. However, HCY partially restored the LDL oxidase activity of redox-copper depleted CP, indicating that the remaining six copper atoms in CP may also be involved in the process. Spectroscopic and oxidation inhibition studies using the Cu(+)-reagent bathocuproine revealed that HCY induced Cu(+)-CP formation, thus promoting its LDL oxidase activity.     

Impact of cofactor on stability of bacterial (CopZ) and human (Atox1) copper chaperones

Here, we present the first characterization of in vitro unfolding and thermodynamic stability of two copper chaperone proteins: Bacillus subtilis CopZ and Homo sapiens Atox1. We find that the unfolding reactions for apo- and Cu(I)-forms of CopZ and Atox1, induced by the chemical denaturant, guanidine hydrochloride (GuHCl), and by thermal perturbation are reversible two-state reactions. For both proteins, the unfolding midpoints shift to higher GuHCl concentrations and the thermodynamic stability is increased in the presence of Cu(I). Despite the same overall fold, apo-CopZ exhibits much lower thermal stability than apo-Atox1. Although the thermal stability of both proteins is increased in the presence of copper, the stabilizing effect is largest for the less stable variant. Divergent energetic properties of the apo- and holo-forms may be linked to conformational changes that facilitate copper transfer to the target.     

Biochemical characterization of AtHMA6/PAA1, a chloroplast envelope Cu(I)-ATPase

Copper is an essential plant micronutrient playing key roles in cellular processes, among them photosynthesis. In Arabidopsis thaliana, copper delivery to chloroplasts, mainly studied by genetic approaches, is thought to involve two P(IB)-type ATPases: AtHMA1 and AtHMA6/PAA1. The lack of biochemical characterization of AtHMA1 and PAA1, and more generally of plant P(IB)-type ATPases, is due to the difficulty of getting high amounts of these membrane proteins in an active form, either from their native environment or after expression in heterologous systems. In this study, we report the first biochemical characterization of PAA1, a plant copper-transporting ATPase. PAA1 produced in Lactococcus lactis is active, forming an aspartyl phosphate intermediate in the presence of ATP and the adequate metal ion. PAA1 can also be phosphorylated using inorganic phosphate in the absence of transition metal. Both phosphorylation types allowed us to demonstrate that PAA1 is activated by monovalent copper ions (and to a lower extent by silver ions) with an apparent affinity in the micromolar range. In agreement with these biochemical data, we also demonstrate that when expressed in yeast, PAA1 induces increased sensitivities to copper and silver. These data provide the first enzymatic characterization of a P(IB-1)-type plant ATPase and clearly identify PAA1 as a high affinity Cu(I) transporter of the chloroplast envelope.     

The affinity of yeast and bacterial SCO proteins for CU(I) and CU(II). A capture and release strategy for copper transfer

SCO (Synthesis of Cytochrome c Oxidase) proteins are present in prokaryotic and eukaryotic cells, and are often required for efficient synthesis of the respiratory enzyme cytochrome c oxidase. The Bacillus subtilis version of SCO (i.e., BsSCO) has much greater affinity for Cu(II) than it does for Cu(I) (Davidson and Hill, 2009), and this has been contrasted to mitochondrial SCO proteins that are characterized as being specific for Cu(I) (Nittis, George and Winge, 2001). This differential affinity has been proposed to reflect the different physiological environments in which these two members of the SCO protein family reside. In this study the affinity of mitochondrial SCO1 from yeast is compared directly to that of BsSCO in vitro. We find that the yeast SCO1 protein has similar preference for Cu(II) over Cu(I), as does BsSCO. We propose a mechanism for SCO function which would involve high-affinity binding to capture Cu(II), and relatively weak binding of Cu(I) to facilitate copper transfer.     

Copper transfer to the N-terminal domain of the Wilson disease protein (ATP7B): X-ray absorption spectroscopy of reconstituted and chaperone-loaded metal binding domains and their interaction with exogenous ligands

The copper-transporting ATPases are 165-175 kDa membrane proteins, composed of 8 transmembrane segments and two large cytosolic domains, the N-terminal copper-binding domain and the catalytic ATP-hydrolyzing domain. In ATP7B, the Wilson disease protein, the N-terminal domain is made up of six metal-binding sub-domains containing the MXCXXC motif which is known to coordinate copper via the two cysteine residues. We have expressed the N-terminal domain of ATP7B as a soluble C-terminal fusion with the maltose binding protein. This expression system produces a protein which can be reconstituted with copper without recourse to the harsh denaturing conditions or low pH reported by other laboratories. Here we describe the reconstitution of the metal binding domains (MBD) with Cu(I) using a number of different protocols, including copper loading via the chaperone, Atox1. X-ray absorption spectra have been obtained on all these derivatives, and their ability to bind exogenous ligands has been assessed. The results establish that the metal-binding domains bind Cu(I) predominantly in a bis cysteinate environment, and are able to bind exogenous ligands such as DTT in a similar fashion to Atox1. We have further observed that exogenous ligand binding induces the formation of a Cu-Cu interaction which may signal a conformational change of the N-terminal domain.     

A novel role for the immunophilin FKBP52 in copper transport

FK506-binding protein 52 (FKBP52) is an immunophilin that possesses peptidylprolyl cis/trans-isomerase (PPIase) activity and is a component of a subclass of steroid hormone receptor complexes. Several recent studies indicate that immunophilins can regulate neuronal survival and nerve regeneration although the molecular mechanisms are poorly understood. To investigate the function of FKBP52 in the nervous system, we employed a yeast two-hybrid strategy using the PPIase domain (domain I) as bait to screen a neonatal rat dorsal root ganglia cDNA expression library. We identified an interaction between FKBP52 domain I and Atox1, a copper-binding metallochaperone. Atox1 interacts with Menkes disease protein and Wilson disease protein (WD) and functions in copper efflux. The interaction between FKBP52 and Atox1 was observed in both glutathione S-transferase pull-down experiments and when proteins were ectopically expressed in human embryonic kidney (HEK) 293T cells and was sensitive to FK506. Interestingly, the FKBP52/Atox1 interaction was enhanced when HEK 293T cells were cultured in copper-supplemented medium and decreased in the presence of the copper chelator, bathocuproine disulfate, suggesting that the interaction is regulated in part by intracellular copper. Overexpression of FKBP52 increased rapid copper efflux in (64)Cu-loaded cells, as did the overexpression of WD transporter. Taken together, our present findings suggest that FKBP52 is a component of the copper efflux machinery, and in so, may also promote neuroprotection from copper toxicity.     

Calbindin-D28K, a 1 alpha,25-dihydroxyvitamin D3-induced calcium-binding protein, binds five or six Ca2+ ions with high affinity

Calbindin-D28K is a 1 alpha,25-dihydroxyvitamin D3-dependent protein that belongs to the superfamily of high affinity calcium-binding proteins which includes parvalbumin, calmodulin, and troponin C. All of these proteins bind Ca2+ ligands by an alpha-helix-loop-alpha-helix domain that is termed an EF-hand. Calbindin-D28K has been reported previously to have four high affinity Ca2(+)-binding sites (KD less than 10(-7)) as quantitated by equilibrium dialysis. With the determination of the amino acid sequence, it was clear that there are in fact six apparent EF-hand domains, although the Ca2(+)-binding functionality of the two additional domains was unclear. It was of interest to quantitate the Ca2(+)-binding ability of chick intestinal calbindin-D28K utilizing several different Ca2+ titration methods that cover a range of macroscopic binding constants for weak or strong Ca2+ sites. Titrations with the Ca2+ chelator dibromo-1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (5,5'-Br2BAPTA), a Ca2+ selective electrode, and as followed by 1H NMR, which measure KD values of 10(-6)-10(-8) M, 10(-4)-10(-7) and 10(-3)-10(-5) M, respectively, gave no evidence for the presence of weak Ca2(+)-binding sites. However, Ca2+ titration of the fluorescent Ca2+ chelator Quin 2 in the presence of calbindin-D28K yielded a least squares fit optimal for 5.7 +/- 0.8 Ca2(+)-binding sites with macroscopic dissociation constants around 10(-8) M. The binding of Ca2+ by calbindin was found to be cooperative with at least two of the sites exhibiting positive cooperativity.     

Silver binding toPseudomonas aeruginosa azurin

Summary The interaction between azurin and silver ions was investigated, by means of ultraviolet, fluorescence and atomic absorption spectroscopies, as a function of the redox state of the protein. The Ag(I) ion has a very low affinity for oxidized azurin. Interestingly, the affinity is much higher for reduced azurin; in this case Ag(I) completely displaces the Cu(I) ion from the native binding site. The effect is very specific for silver ions since other ions, such as Hg(II), Ni(II) and Cd(II), do not produce the same effect. Treatment of reduced and oxidized azurin with excess Ag(I) (2-8-fold stoichiometric) shows that there is a second binding site for silver ions on the protein which can also bind Cu(II) and Hg(II) with comparable affinities.