Metal ions binding to NAD-glycohydrolase from the venom of Agkistrodon acutus: regulation of multicatalytic activity

AA-NADase from Agkistrodon acutus venom is a unique multicatalytic enzyme with both NADase and AT(D)Pase activities. Among all identified NADases, only AA-NADase contains Cu(2+) ions that are essential for its multicatalytic activity. In this study, the interactions between divalent metal ions and AA-NADase and the effects of metal ions on its structure and activity have been investigated by equilibrium dialysis, isothermal titration calorimetry, fluorescence, circular dichroism, dynamic light scattering and HPLC. The results show that AA-NADase has two classes of Cu(2+) binding sites, one activator site with high affinity and approximately six inhibitor sites with low affinity. Cu(2+) ions function as a switch for its NADase activity. In addition, AA-NADase has one Mn(2+) binding site, one Zn(2+) binding site, one strong and two weak Co(2+) binding sites, and two strong and six weak Ni(2+) binding sites. Metal ion binding affinities follow the trend Cu(2+) > Ni(2+) > Mn(2+) > Co(2+) > Zn(2+), which accounts for the existence of one Cu(2+) in the purified AA-NADase. Both NADase and ADPase activities of AA-NADase do not have an absolute requirement for Cu(2+), and all tested metal ions activate its NADase and ADPase activities and the activation capacity follows the trend Zn(2+) > Mn(2+) > Cu(2+) ~Co(2+) > Ni(2+). Metal ions serve as regulators for its multicatalytic activity. Although all tested metal ions have no obvious effects on the global structure of AA-NADase, Cu(2+)- and Zn(2+)-induced conformational changes around some Trp residues have been observed. Interestingly, each tested metal ion has a very similar activation of both NADase and ADPase activities, suggesting that the two different activities probably occur at the same site.     

Structural basis for Ni(2+) transport and assembly of the urease active site by the metallochaperone UreE from Bacillus pasteurii

Bacillus pasteurii UreE (BpUreE) is a putative chaperone assisting the insertion of Ni(2+) ions in the active site of urease. The x-ray structure of the protein has been determined for two crystal forms, at 1.7 and 1.85 A resolution, using SIRAS phases derived from a Hg(2+)-derivative. BpUreE is composed of distinct N- and C-terminal domains, connected by a short flexible linker. The structure reveals the topology of an elongated homodimer, formed by interaction of the two C-terminal domains through hydrophobic interactions. A single Zn(2+) ion bound to four conserved His-100 residues, one from each monomer, connects two dimers resulting in a tetrameric BpUreE known to be formed in concentrated solutions. The Zn(2+) ion can be replaced by Ni(2+) as shown by anomalous difference maps obtained on a crystal of BpUreE soaked in a solution containing NiCl(2). A large hydrophobic patch surrounding the metal ion site is surface-exposed in the biologically relevant dimer. The BpUreE structure represents the first for this class of proteins and suggests a possible role for UreE in the urease nickel-center assembly.     

A theoretical investigation of the functional role of the axial methionine ligand of the Cu(A) site in cytochrome c oxidase

The functional roles of the amino acid residues of the Cu(A) site in bovine cytochrome c oxidase (CcO) were investigated by utilizing hybrid quantum mechanics (QM)/molecular mechanics (MM) calculations. The energy levels of the molecular orbitals (MOs) involving Cu d(zx) orbitals unexpectedly increased, as compared with those found previously with a simplified model system lacking the axial Met residue (i.e., Cu(2)S(2)N(2)). This elevation of MO energies stemmed from the formation of the anti-bonding orbitals, which are generated by hybridization between the d(zx) orbitals of Cu ions and the p-orbitals of the S and O atoms of the axial ligands. To clarify the roles of the axial Met ligand, the inner-sphere reorganization energies of the Cu(A) site were computed, with the Met residue assigned to either the QM or MM region. The reorganization energy slightly increased when the Met residue was excluded from the QM region. The existing experimental data and the present structural modeling study also suggested that the axial Met residue moderately increased the redox potential of the Cu(A) site. Thus, the role of the Met may be to regulate the electron transfer rate through the fine modulation of the electronic structure of the Cu(A) "platform", created by two Cys/His residues coordinated to the Cu ions. This regulation would provide the optimum redox potential/reorganization energy of the Cu(A) site, and thereby facilitate the subsequent cooperative reactions, such as the proton pump and the enzymatic activity, of CcO. This article is part of a Special Issue entitled: Allosteric cooperativity in respiratory proteins.     

The structure of sitting-atop complexes of metalloporphyrins studied by theoretical methods

The metallation of tetrapyrroles is believed to proceed via a sitting-atop (SAT) complex, in which some of the pyrrole nitrogen atoms are still protonated and the metal ion resides above the ring plane. No crystal structure of such a complex has been presented, but NMR and extended X-ray absorption fine structure (EXAFS) data has been reported for Cu(2+) in acetonitrile. We have used density functional calculations to obtain reasonable models for SAT complexes of porphyrins with Mg(2+), Fe(2+), and Cu(2+). The results show that there are many possible SAT complexes with 1-5 solvent molecules, one or two metal ions, and cis or trans protonation of the porphyrin ring. Many of these have similar energies and their relative stabilities vary with the metal ion. A complex with two cis pyrrolenine nitrogens atoms and 2-4 solvent molecules coordinated to Cu(2+) fits the NMR and EXAFS data best. However, we cannot fully exclude the possibility that what is observed is rather a mixture of a doubly protonated porphyrin and the copper porphyrin. Mg(2+) has a lower affinity for porphyrin and stronger affinity for water, so a complex with five water molecules and only one bond to porphyrin seems to be most stable. For Fe(2+), a cis structure with two first-sphere water molecules and four interactions to the porphyrin seems to be most likely.     

Alzheimer's disease amyloid-beta binds copper and zinc to generate an allosterically ordered membrane-penetrating structure containing superoxide dismutase-like subunits

Amyloid beta peptide (Abeta) is the major constituent of extracellular plaques and perivascular amyloid deposits, the pathognomonic neuropathological lesions of Alzheimer's disease. Cu(2+) and Zn(2+) bind Abeta, inducing aggregation and giving rise to reactive oxygen species. These reactions may play a deleterious role in the disease state, because high concentrations of iron, copper, and zinc have been located in amyloid in diseased brains. Here we show that coordination of metal ions to Abeta is the same in both aqueous solution and lipid environments, with His(6), His(13), and His(14) all involved. At Cu(2+)/peptide molar ratios >0.3, Abeta coordinated a second Cu(2+) atom in a highly cooperative manner. This effect was abolished if the histidine residues were methylated at N(epsilon)2, indicating the presence of bridging histidine residues, as found in the active site of superoxide dismutase. Addition of Cu(2+) or Zn(2+) to Abeta in a negatively charged lipid environment caused a conformational change from beta-sheet to alpha-helix, accompanied by peptide oligomerization and membrane penetration. These results suggest that metal binding to Abeta generated an allosterically ordered membrane-penetrating oligomer linked by superoxide dismutase-like bridging histidine residues.     

Copper alters aggregation behavior of prion protein and induces novel interactions between its N- and C-terminal regions

Copper is reported to promote and prevent aggregation of prion protein. Conformational and functional consequences of Cu(2+)-binding to prion protein (PrP) are not well understood largely because most of the Cu(2+)-binding studies have been performed on fragments and truncated variants of the prion protein. In this context, we set out to investigate the conformational consequences of Cu(2+)-binding to full-length prion protein (PrP) by isothermal calorimetry, NMR, and small angle x-ray scattering. In this study, we report altered aggregation behavior of full-length PrP upon binding to Cu(2+). At physiological temperature, Cu(2+) did not promote aggregation suggesting that Cu(2+) may not play a role in the aggregation of PrP at physiological temperature (37 °C). However, Cu(2+)-bound PrP aggregated at lower temperatures. This temperature-dependent process is reversible. Our results show two novel intra-protein interactions upon Cu(2+)-binding. The N-terminal region (residues 90-120 that contain the site His-96/His-111) becomes proximal to helix-1 (residues 144-147) and its nearby loop region (residues 139-143), which may be important in preventing amyloid fibril formation in the presence of Cu(2+). In addition, we observed another novel interaction between the N-terminal region comprising the octapeptide repeats (residues 60-91) and helix-2 (residues 174-185) of PrP. Small angle x-ray scattering studies of full-length PrP show significant compactness upon Cu(2+)-binding. Our results demonstrate novel long range inter-domain interactions of the N- and C-terminal regions of PrP upon Cu(2+)-binding, which might have physiological significance.     

Thermodynamics and fluorescence studies of the interactions of cyclooctapeptides with Hg2+, Pb2+, and Cd2+

The purpose of this work is to characterize the interactions of cyclooctapeptides (CP) containing glutamyl and/or cysteinyl residues with common heavy-metal ions in order to facilitate the design of cyclopeptides as sensors for metal ions. Isothermal titration calorimetry studies show that cyclooctapeptides containing glutamyl and/or cysteinyl residues bind these Hg(2+) and Pb(2+) over Cd(2+) and other common metal ions. Differential binding isotherms, in their interactions with Hg(2+), support a two-binding site model, whereas pertinent interactions with Pb(2+) support a 2:1 stoichiometry, suggesting a CP/Pb(2+)/CP mode of complexation. The cyclooctapeptide containing both glutamyl and cysteinyl residues shows a significant binding affinity for Hg(2+) (K(a)=7.6x10(7)M(-1)), which is both enthalpically and entropically driven. The fluorescence of these cyclooctapeptides showed pronounced fluorescence quenching responses to Hg(2+) over Pd(2+) and Cd(2+). Stern-Volmer analyses of the dependence of fluorescence intensity on Hg(2+) and Pb(2+) are reported. The observed trends are useful for the design of Hg(2+) sensors based on fluorophore-tagged cyclooctapeptides.     

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.     

Metal-induced point defects in DNA: model and mechanisms

The aim of this work was to study the role of H3O+ and transition-metal (TM) ions in keto-enol and amino-imino tautomeric transitions in DNA base pairs and depurination. In this regard, we discuss the thermodynamic model of ion-DNA interactions and UV display of double-proton transfer (DPT) in GC. The probabilities and energies of rare tautomeric forms of GC pairs in DNA induced by H3O+ and TMwere determined being in the range from0.02 (forMg2+) to 1 ( forCu2+), and from 0 kcal/m (for Cu2+) to 2.3 kcal/m (for Mg2+), respectively. It was shown that 3'ACC5'/5'TGG3' site of DNA double helix, which corresponds to the only triplet 5'UGG3' of RNA that codes the most valuable amino acid tryptophan, is a good target for TM ions to attack. It was also shown that the only way to obtain the tryptophan-coding 5'UGG3' triplet in RNA via transition-type G --> A point mutation caused by TM ions is their interaction with the site of a DNA double helix, which corresponds to 5'CGG3' triplet of RNA that codes arginine.     

Equilibrium and kinetics of biosorption of cadmium(II) and copper(II) ions by wheat straw

Biosorption equilibrium and kinetics of Cd(2+) and Cu(2+) ions on wheat straw, Triticum aestivum, in an aqueous system were investigated. Among the models tested, namely the Langmuir, Freundlich, Temkin, and Dubinin-Radushkevich isotherms, the biosorption equilibrium for both Cd(2+) and Cu(2+) was best described by the Langmuir model. The Langmuir biosorption capacity for Cd(2+) was about 27% higher than that for Cu(2+). It was also found that biosorption of Cd(2+) and Cu(2+) by wheat straw followed second-order kinetics. The equilibrium amount of metal ions adsorbed onto the wheat straw increased with increasing of pH from 4.0 to 7.0, and the effect was more pronounced for Cd(2+) than for Cu(2+). The equilibrium adsorbed amount also increased with the initial concentration of the metal ions, as expected. On the other hand, an increase of temperature from 25 to 30 degrees C only enhanced the biosorption of Cd(2+) and Cu(2+) slightly. The apparent temperature independence and the strong pH dependence of the amount of metal ions adsorbed along with moderate mean free energies of biosorption (between 8.0 and 12.9 kJ mol(-1)) altogether indicate that biosorption of Cd(2+) and Cu(2+) by wheat straw might follow a chemisorption mechanism.     

Wheat germ agglutinin and other selected lectins increase synthesis of decay-accelerating factor in human endothelial cells.

Decay accelerating factor (DAF) is a cell-surface phosphatidylinositol-anchored protein that protects the cell from inadvertent complement attack by binding to and inactivating C3 and C5 convertases. We have measured DAF on human umbilical vein endothelial cells (HUVEC) by immunoradiometric assay after its removal by phosphatidylinositol-specific phospholipase C or Nonidet P-40 detergent extraction and have previously demonstrated that DAF synthesis can be stimulated by phorbol ester activation of protein kinase C. We now report that although stimulation (4-48 h) of HUVEC with various cytokines, including TNF, IL-1, and IFN-gamma, did not alter DAF levels, wheat germ agglutinin (WGA) (5-50 micrograms/ml), a lectin specific for binding N-acetyl neuraminic acid and N-acetyl glucosamine residues, increased DAF levels fivefold when incubated with HUVEC for 12 to 24 h. The lectins Con A and PHA also stimulated DAF expression twofold, whereas a number of others including Ulex europaeus, Bandeiraea simplicifolia lectin I, and Ricinus communis agglutinin I, which bind to endothelial cells, were inactive. The increase in DAF by WGA was inhibited by N-acetyl glucosamine (10-50 mM) but by neither N-acetyl neuraminic acid nor removal of surface N-acetyl neuraminic acid with neuraminidase. However, succinylated WGA, which has unaltered affinity for N-acetyl glucosamine but not longer binds N-acetyl neuraminic acid, was inactive. These data suggest that the binding of WGA to sugar residues alone is not sufficient to trigger DAF expression and that occupation of additional, specific sites are required. The increase in DAF levels on HUVEC was blocked by inhibitors of RNA and protein synthesis. We conclude that continuous occupation by WGA of specific binding sites on HUVEC triggers events leading to DAF synthesis. This unique, long term stimulation of endothelial cells by lectins may be relevant to cell:cell interactions at the endothelium.     

Metal ion binding to human hemopexin

Binding of divalent metal ions to human hemopexin (Hx) purified by a new protocol has been characterized by metal ion affinity chromatography and potentiometric titration in the presence and absence of bound protoheme IX. ApoHx was retained by variously charged metal affinity chelate resins in the following order: Ni(2+) > Cu(2+) > Co(2+) > Zn(2+) > Mn(2+). The Hx-heme complex exhibited similar behavior except the order of retention of the complex on Zn(2+)- and Co(2+)-charged columns was reversed. One-dimensional (1)H NMR of apoHx in the presence of Ni(2+) implicates at least two His residues and possibly an Asp, Glu, or Met residue in Ni(2+) binding. Potentiometric titrations establish that apoHx possesses more than two metal ion binding sites and that the capacity and/or affinity for metal ion binding is diminished when heme binds. For most metal ions that have been studied, potentiometric data did not fit to binding isotherms that assume one or two independent binding sites. For Mn(2+), however, these data were consistent with a high-affinity site [K(A) = (15 +/- 3) x 10(6) M(-)(1)] and a low-affinity site (K(A) 相似文献   

The whole hexapeptide repeats domain from avian PrP displays untypical hallmarks in aspect of the Cu2+ complexes formation

Prions, the infectious agents responsible for the transmissible spongiform encephalopathies (TSEs) have defied full characterization for decades. Although the interactions of Cu(2+) ions with PrP both in vivo and in vitro are well documented, there are still a lot of ambiguities concerning the biological and chemical nature of these effects. In this work, we have investigated the interactions of Cu(2+) ions with whole repeat region of the copper-binding domain (hexapeptide repeats) of chicken PrP. Our results provide explanations for the structural and chemical basis of the specific interactions of Cu(2+) ions with the hexapeptide repeat region. Furthermore, we show that SOD-like activity depends on Cu(2+) complexes.     

Calbindin D(9k): a protein optimized for calcium binding at neutral pH.

The binding of calcium ions by EF-hand proteins depends strongly on the electrostatic interactions between Ca(2+) ions and negatively charged residues of these proteins. We have investigated the pH dependence of the binding of Ca(2+) ions by calbindin D(9k). This protein offers a unique possibility for interpretation of such data since the pK(a) values of all ionizable groups are known. The binding is independent of pH between 7 and 9, where maximum calcium affinity is observed. An abrupt decrease in the binding affinity is observed at pH values below 7. This decrease is due to protonation of acidic groups, leading to modification of protein charges. The pH dependence of the product of the two macroscopic Ca(2+)-binding constants can be formally described by the involvement of two acidic groups with pK(a) = 6.6. Monte Carlo calculations show that the reduction of Ca(2+) binding is strictly determined by variable electrostatic interactions due to pH-dependent changes not only in the binding sites, but also of the overall charge of the protein.     

Investigation of human erythrocyte superoxide dismutase by 1H nuclear-magnetic-resonance spectroscopy.

The 170MHZ 1 H n.m.r. spectra of the Cu(II)/Zn(II), Cu(I)/Zn(II) and apo- forms of human erythrocyte superoxide dismutase (EC 1.15.1.1) are reported. Resonances are assigned to the C-2 and C-4 protons of histidine residues in the active site, and it is suggested that five or six histidine residues serve as ligands to the metal ions in each subunit of the enzyme. The remaining assigned resonances are associated with histidine-41, N-terminal N-acetyl group, histidine- 108 and cysteine- 109. A comparison of the n.m.r. spectra of human and bovine superoxide dismutases suggests significant structural homology.     

Binding of divalent metal ions to calcium-free peroxidase: thermodynamic and kinetic studies

Thermodynamics of binding of divalent metal ions including Ca(2+) , Mg(2+) , Ba(2+) , and Cd(2+) to Ca-free horseradish peroxidase (HRP) enzyme was investigated using UV/VIS spectrophotometry and molecular-mechanic (MM) calculations. According to the obtained binding and thermodynamic parameters, trend of the relative binding affinities of these divalent metal cations was found to be: Ca(2+) >Cd(2+) >Mg(2+) >Ba(2+) . Binding analysis based on Scatchard and Hill models showed positive cooperativity effect between the two distal and proximal binding sites. Furthermore, kinetics of binding and reconstitution process was examined (using relaxation-time method) for binding of Ca(2+) (as the typical metal ion) to Ca-free HRP, which was found a second-order type having a two-step mechanism involving fast formation of Ca-free HRP/1?Ca(2+) as the kinetic intermediate in step 1. Finally, by means of MM calculations, the comparative stability energies were evaluated for binding of M(2+) metal cations to Ca-free HRP. Based on MM calculations, preferential binding of Ca(2+) ion was occurred on distal and proximal binding sites of Ca-free HRP associated with higher stability energies (E(total) ). Indeed, among the divalent metal ions, Ca(2+) with the highest binding affinity (maximum value of K(bin) and minimum value of ΔG$\rm{{_{bin}^{0}}}$), maximum value of exothermic binding enthalpy, and stability energies stabilizes the HRP structure along with an optimized catalytic activity.     

Effects of divalent metal ions on the alphaB-crystallin chaperone-like activity: spectroscopic evidence for a complex between copper(II) and protein

AlphaB-crystallin is a small heat shock protein, showing chaperone-like activity, that is expressed in the lens and in several other tissues. The role of some metal ions in the alphaB-crystallin biology starts to be well documented. In some neuro-degenerative pathologies, like Parkinson and Alzheimer's diseases, alphaB-crystallin is expressed at high levels. In the same pathologies an accumulation of divalent metal cations is observed. In order to investigate the interactions between human alphaB-crystallin and divalent metal ions, the effect of copper, zinc and calcium on the chaperone-like activity of the protein has been studied. Copper and zinc at concentrations 0.1 and 1 mM significantly increase the chaperone-like activity, whereas calcium 1 mM completely inhibits activity. Electron paramagnetic resonance (EPR) and circular dichroism (CD) spectra indicate the possible complex formation between Cu(II) and protein at physiological pH. Molecular modeling calculations, carried out for the probable Cu(II) binding site, suggest that a complex with three histidine residues is possible.     

Specific DNA cleavage by an analog of netropsin containing a copper(II) chelating peptide Gly-Gly-His]

Experimental data are reported on DNA-cleaving activity of the synthetic netropsin analogs consisting of the two N-propylpyrrole carboxamide units linked covalently through two or three glycine residues to a copper-chelating tripeptide glycyl-glycyl-L-histidine. Incubation of DNA restriction fragment and netropsin analog in the presence of ascorbate, hydrogen peroxide and Cu2+ ions resulted in selective cleavage of the DNA at or near the preferred sites for binding of netropsin analog. A similar cleavage pattern is observed after X-ray irradiation of DNA complexes with netropsin analogs tethered with Cu2+ ions. The cleavage patterns are found to be dependent on the length of the connecting chain between the histidine-containing tripeptide and netropsin analog. The netropsin analog containing three glycine residues in the connecting chain, but not the analog with a shorter linker chain, can generate an intense cleavage of one of the two polynucleotide chains at a position corresponding to the presumed binding site for the dimeric ligand species. More than 50% of the total DNA can be cleaved at this position after X-ray irradiation. From analysis of the nucleotide sequences surrounding the preferred cleavage site on several DNA fragments we found that the consensus is 5'-TTTTNCA*AAA-3', where N is an arbitrary nucleotide. The Cu(2+)-mediated cleavage of DNA occurs at the second adenine (indicated by an asterisk) from the 5'-end of the sequence. The greatest cleavage activity is observed when the molar ratio of Cu2+ to the netropsin analog is equal to 0.5. Evidently, the Cu(2+)-ligated and unligated oligopeptide species interacts with each other to form a heterodimer bound to DNA at the cleavage site. To test the validity of this model we have studied the binding of unligated netropsin analog and netropsin analog complexed with Cu2+ ion to a self-complementary oligonucleotide 5'-GCGTTTTGCAAAACGC-3'. It is found that binding of Cu(2+)-ligated netropsin analog to the DNA oligomer preincubated with unligated form of the oligopeptide is a cooperative process for which interactions between the two bound ligands are responsible. The cooperativity parameter is estimated to be on the order of factor 6. Finally, a model is proposed in which a heterodimer stabilized by interligand beta-sheet binds in the minor DNA groove.     

A plasma membrane-associated protein of Arabidopsis thaliana AtPCaP1 binds copper ions and changes its higher order structure

PCaP1, a hydrophilic cation-binding protein, is bound to the plasma membrane in Arabidopsis thaliana. We focused on the physicochemical properties of PCaP1 to understand its uniqueness in terms of structure and binding of metal ions. On fluorescence analysis, PCaP1 showed a signal of structural change in the presence of Cu(2+). The near-UV CD spectra showed a marked change of PCaP1 in CuCl(2) solution. The far-UV CD spectra showed the presence of alpha-helices and the intrinsically unstructured region. However, addition of Cu(2+) gave no change in the far-UV CD spectra. These results indicate that Cu(2+) induced a change in the tertiary structure without changing the secondary structure. The protein was sensitive to proteinase in the presence of Cu(2+), supporting that Cu(2+) is involved in the structural change. The PCaP1 solution was titrated with CuCl(2) and the change in the fluorescence spectrum was monitored to characterize Cu(2+)-binding properties. The obtained values of K(d) for Cu(2+) and the ligand-binding number were 10 microM and six ions per molecule, respectively. These findings indicate that PCaP1 has a high Cu(2+)-binding capacity with a relatively high affinity. PCaP1 lacks cysteine and histidine residues. A large number of glutamate residues may be involved in the Cu(2+) binding.