Copper Binding Site Research Articles

Site-Specific Interactions of Cu(II) with α and β-Synuclein: Bridging the Molecular Gap between Metal Binding and Aggregation

The aggregation of alpha-synuclein (AS) is a critical step in the etiology of Parkinson's disease (PD) and other neurodegenerative synucleinopathies. Protein-metal interactions play a critical role in AS aggregation and might represent the link between the pathological processes of protein aggregation and oxidative damage. Our previous studies established a hierarchy in AS-metal ion interactions, where Cu(II) binds specifically to the protein and triggers its aggregation under conditions that might be relevant for the development of PD. In this work, we have addressed unresolved structural details related to the binding specificity of Cu(II) through the design of site-directed and domain-truncated mutants of AS and by the characterization of the metal-binding features of its natural homologue beta-synuclein (BS). The structural properties of the Cu(II) complexes were determined by the combined application of nuclear magnetic resonance, electron paramagnetic resonance, UV-vis, circular dichroism spectroscopy, and matrix-assisted laser desorption ionization mass spectrometry (MALDI MS). Two independent, noninteracting copper-binding sites with significantly different affinities for the metal ion were detected in the N-terminal regions of AS and BS. MALDI MS provided unique evidence for the direct involvement of Met1 as the primary anchoring residue for Cu(II) in both proteins. Comparative spectroscopic analysis of the two proteins allowed us to deconvolute the Cu(II) binding modes and unequivocally assign the higher-affinity site to the N-terminal amino group of Met1 and the lower-affinity site to the imidazol ring of the sole His residue. Through the use of competitive chelators, the affinity of the first equivalent of bound Cu(II) was accurately determined to be in the submicromolar range for both AS and BS. Our results prove that Cu(II) binding in the C-terminal region of synucleins represents a nonspecific, very low affinity process. These new insights into the bioinorganic chemistry of PD are central to an understanding of the role of Cu(II) in the fibrillization process of AS and have implications for the molecular mechanism by which BS might inhibit AS amyloid assembly.

Both Met(109) and Met(112) are utilized for Cu(II) coordination by the amyloidogenic fragment of the human prion protein at physiological pH

The prion protein is a ubiquitous neuronal membrane protein. Misfolding of the prion protein has been implicated in transmissible spongiform encephalopathies (prion diseases). It has been demonstrated that the human prion protein (PrP) is capable of coordinating at least five CuII ions under physiological conditions; four copper binding sites can be found in the octarepeat domain between residues 61 and 91, while another copper binding site can be found in the unstructured “amyloidogenic” domain between residues 91 and 126 PrP(91–126). Herein we expand upon a previous study [J. Shearer, P. Soh, Inorg. Chem. 46 (2007) 710–719] where we demonstrated that the physiologically relevant high affinity CuII coordination site within PrP(91–126) is found between residues 106 and 114. It was shown that CuII is contained within a square planar (N/O)3S coordination environment with one His imidazole ligand (H(111)) and one Met thioether ligand (either M(109) or M(112)). The identity of the Met thioether ligand was not identified in that study. In this study we perform a detailed investigation of the CuII coordination environment within the PrP fragment containing residues 106–114 (PrP(106–114)) involving optical, X-ray absorption, EPR, and fluorescence spectroscopies in conjunction with electronic structure calculations. By using derivatives of PrP(106–114) with systematic Met→Ile “mutations” we show that the CuII coordination environment within PrP(106–114) is actually comprised of a mixture of two major species; one CuII(N/O)3S center with the M(109) thioether coordinated to CuII and another CuII(N/O)3S center with the M(112) thioether coordinated to CuII. Furthermore, deletion of one or more Met residues from the primary sequence of PrP(106–114) both reduces the CuII affinity of the peptide by two to seven fold, and renders the resulting CuII metallopeptides redox inactive. The biological implications of these findings are discussed.

One heme, diverse functions: using biosynthetic myoglobin models to gain insights into heme-copper oxidases and nitric oxide reductases.

The heme-copper oxidase superfamily: Heme-copper oxidase (HCO) and nitric oxide reductase (NOR) Heme-copper oxidases (HCOs) are a superfamily of terminal oxidases present in the respiratory chains of both bacteria and eukaryotic mitochondria.5 HCOs utilize O2 as a terminal electron acceptor, catalyzing the four electron reduction of O2 to H2O (O2 + 4e− + 8H+in → 2H2O + 4H+out), while generating a proton gradient that is used to drive the production of ATP. Nitric oxide reductase (NOR) is a metalloenzyme in the denitrification pathway responsible for the two electron reduction of NO to N2O (2NO + 2H+ + 2e− → N2O + H2O), a reaction that is nonelectrogenic (i.e., no charge translocation across the membrane).6,7 Denitrifying NO reductases are also members of the heme-copper oxidase superfamily. However, despite the diversity of their native functions, NOR and HCO have cross-reactivity; NOR is capable of reducing O2 and HCO is capable of reducing NO, although less efficiently.8–10 Not all members of the HCO superfamily are capable of NO reduction. The oxidase from bovine heart has no NO activity.11 In contrast, the ba3 and caa3 type oxidases from Thermus thermophilus9 and the cbb3 type oxidase from Pseudomonas stutzeri10 are capable of catalytically reducing NO to N2O, with the cbb3 oxidase being ~2 orders of magnitude faster than their oxidase counterparts. Although X-ray crystal structures are available5 for the oxidase from bovine heart,12,13 the aa3 type from Paracoccus denitrificans14,15 and Rhodobacter sphaeroides,16 the ba3 type from Thermus thermophilus,17 and the bo3 type ubiquinol oxidase from Escherichia coli,18 there is still no crystal structure for the cbb3 type oxidase, which from sequence alignments are predicted to be the most structurally similar oxidase to NOR.19 These crystal structures show the catalytic core of HCOs to contain a low-spin heme center and a high-spin heme/CuB binuclear center, (cytochrome c oxidase, CcO, also a member of the HCO superfamily, contains an additional binuclear CuA center). The CuB center consists of three strictly conserved His which bind a central Cu ion. One of these His is covalently attached to a strictly conserved Tyr to form a His-Tyr crosslink that is thought to play both a structural and functional role in O2 reduction.20 Another important characteristic of the heme-copper active site is the spin-coupled nature of the center in the oxidized or met form (i.e., CuII-FeIII) of the protein. Spin-coupling is thought to be provided by a bridging ligand between the two metal centers.21–25 Currently, no crystal or solution structure exists for bacterial NOR but sequence alignments and structural modeling have shown NORs to be structurally homologous to subunit I of HCOs.6,7 All six His metal ligands to the active site of HCO (two to the low-spin heme, one to the high-spin heme and three to CuB) are conserved in NOR at the same predicted positions in the 12 trans-membrane helices of HCO. The major structural difference between HCO and NOR is the replacement of a copper binding site (CuB) with a non-heme iron binding site (FeB) in NOR. Additionally, several conserved glutamates have been identified in the vicinity of the catalytic heme/non-heme FeB site of NOR which are predicted to play a role in iron binding and/or catalysis.26 Because of the importance of HCO to respiration, the structural similarities between NOR and HCO, and the importance of NOR to the denitrification cycle, it is important to reach a clear understanding of the structure and function of these vital enzymes. Biochemical, spectroscopic, and X-ray crystallographic studies of both HCOs12–15,27–37 and NORs38–49 are sometimes hampered by their size and complexity. Both are membrane-bound proteins containing several different metal cofactors, making their isolation difficult and spectroscopic characterization complicated (e.g., UV-vis and MCD spectra of the heme-copper center in HCO are masked by the low-spin heme center). These difficulties call upon the need for innovative ways to gain insight into the structure-function relationship of these heme proteins.

Investigation of the copper binding site and the role of histidine as a ligand in riboflavin binding protein.

Riboflavin Binding Protein (RBP) binds copper in a 1:1 molar ratio, forming a distinct well-ordered type II site. The nature of this site has been examined using X-ray absorption and pulsed electron paramagnetic resonance (EPR) spectroscopies, revealing a four coordinate oxygen/nitrogen rich environment. On the basis of analysis of the Cambridge Structural Database, the average protein bound copper-ligand bond length of 1.96 A, obtained by extended x-ray absorption fine structure (EXAFS), is consistent with four coordinate Cu(I) and Cu(II) models that utilize mixed oxygen and nitrogen ligand distributions. These data suggest a Cu-O 3N coordination state for copper bound to RBP. While pulsed EPR studies including hyperfine sublevel correlation spectroscopy and electron nuclear double resonance show clear spectroscopic evidence for a histidine bound to the copper, inclusion of a histidine in the EXAFS simulation did not lead to any significant improvement in the fit.

Interaction of Pseudomonas putida CZ1 with clays and ability of the composite to immobilize copper and zinc from solution

The present study was conducted to determine the abilities of the living and nonliving Pseudomonas putida CZ1 cells, clays (goethite, kaolinite, smectite and manganite) and their composites to accumulate copper and zinc from a liquid medium, and elucidate the role of microbes on the mobility of heavy metals. Various mixtures of bacteria and clays were exposed to solutions of 0.025 mM or 0.5 mM Cu(II) and Zn(II) in 0.01 M KNO 3 to differentiate between so-called “high-affinity” sites and “low-affinity” sites. Clays associated in an edge-on orientation to the cells was observed by electron microscope (EM) examination of these metal-treated bacteria–clay aggregates. Adsorption experiments and desorption with 1.0 M CH 3COOK solution indicated that clays contain more high-affinity copper binding sites and less high-affinity zinc binding sites than that of bacteria, however, bacteria are involved in more low-affinity heavy-metal-binding sites. Carboxyl group activity is more important at weak-binding sites than at strong-binding sites. TEM-EDS analysis confirmed that most of Zn removed from solution was associated with P. putida CZ1 in the composites. These results suggest that bacteria play an important role in regulating the mobility of heavy metals in the soil environment.

Copper and zinc binding properties of the N-terminal histidine-rich sequence of Haemophilus ducreyi Cu,Zn superoxide dismutase

The Cu,Zn superoxide dismutase (Cu,ZnSOD) isolated from Haemophilus ducreyi possesses a His-rich N-terminal metal binding domain, which has been previously proposed to play a copper(II) chaperoning role. To analyze the metal binding ability and selectivity of the histidine-rich domain we have carried out thermodynamic and solution structural analysis of the copper(II) and zinc(II) complexes of a peptide corresponding to the first 11 amino acids of the enzyme (H(2)N-HGDHMHNHDTK-OH, L). This peptide has highly versatile metal binding ability and provides one and three high affinity binding sites for zinc(II) and copper(II), respectively. In equimolar solutions the MHL complexes are dominant in the neutral pH-range with protonated lysine epsilon-amino group. As a consequence of its multidentate nature, L binds zinc and copper with extraordinary high affinity (K(D,Zn)=1.6x10(-9)M and K(D,Cu)=5.0x10(-12)M at pH 7.4) and appears as the strongest zinc(II) and copper(II) chelator between the His-rich peptides so far investigated. These K(D) values support the already proposed role of the N-terminal His-rich region of H. ducreyi Cu,ZnSOD in copper recruitment under metal starvation, and indicate a similar function in the zinc(II) uptake, too. The kinetics of copper(II) transfer from L to the active site of Cu-free N-deleted H. ducreyi Cu,ZnSOD showed significant pH and copper-to-peptide ratio dependence, indicating specific structural requirements during the metal ion transfer to the active site. Interestingly, the complex CuHL has significant superoxide dismutase like activity, which may suggest multifunctional role of the copper(II)-bound N-terminal His-rich domain of H. ducreyi Cu,ZnSOD.

An Oxidized Tryptophan Facilitates Copper Binding in Methylococcus capsulatus-secreted Protein MopE

Proteins can coordinate metal ions with endogenous nitrogen and oxygen ligands through backbone amino and carbonyl groups, but the amino acid side chains coordinating metals do not include tryptophan. Here we show for the first time the involvement of the tryptophan metabolite kynurenine in a protein metal-binding site. The crystal structure to 1.35 angstroms of MopE* from the methane-oxidizing Methylococcus capsulatus (Bath) provided detailed information about its structure and mononuclear copper-binding site. MopE* contains a novel protein fold of which only one-third of the structure displays similarities to other known folds. The geometry around the copper ion is distorted tetrahedral with one oxygen ligand from a water molecule, two histidine imidazoles (His-132 and His-203), and at the fourth distorted tetrahedral position, the N1 atom of the kynurenine, an oxidation product of Trp-130. Trp-130 was not oxidized to kynurenine in MopE* heterologously expressed in Escherichia coli, nor did this protein bind copper. Our findings indicate that the modification of tryptophan to kynurenine and its involvement in copper binding is an innate property of M. capsulatus MopE*.

Aceruloplasminemia: a case report

Hereditary aceruloplasminemia is a rare autosomal recessive disease, firstly identified by Miyajima et al. in Japan in 1987 [1]. The disease is caused by the absence of an a2glycoprotein, the ceruloplasmin (Cp), a copper-containing ferroxidase, mainly synthesized in hepatocytes and widely expressed, including the central nervous system, which catalyses the oxidation of ferrous to ferric iron, a change required for release of iron to plasma transferrin [2]. It is hypothesized that in reticuloendothelial (RE) cells and hepatocytes Cp cooperates to export iron with the iron exporter protein ferroportin 1 (FPN1) [3]. As a consequence, Cp deficiency results in iron deposition in the liver, pancreas, basal ganglia, and other organs [2]. The Cp gene maps to chromosome 3q21–24 [4], consists of 19 exons [5] and encodes a protein of 1,046 amino acids [6]. Recently 20th exon has been recognized as the result of a brain-specific alternative splicing. This adds to the protein an aminoacid sequence that makes Cp a glycosylphosphatidylinositol (GPI)-anchored protein in the brain [7]. Under normal circumstances, infact, circulating Cp does not cross the blood–brain barrier. Through the oxidation mediated by GPI-linked Cp expressed in the astrocytes, iron could be taken from the circulation, incorporated into transferring derived from oligodendrocytes and then transferred to the neuronal cells [2]. Ceruloplasmin deficiency is a characteristic feature in copper metabolic disorders, including Wilson’s disease and Menkes’ disease. In Wilson’s disease, an inability to transfer copper into the Cp precursor protein, apoceruloplasmin, and a decrease in biliary copper excretion results in serum ceruloplasmin deficiency and excess copper accumulation. In Menkes’ disease, copper absorption from the intestine is decreased, leading to copper and Cp deficiencies in the body. In contrast, aceruloplasminemia, is an iron metabolic disorder in which ceruloplasmin deficiency is caused by a lack of apoceruloplasmin biosynthesis and copper metabolism is not disturbed. In Wilson’s disease and Menkes’ disease, several gene mutations in the coppertransporting ATPases (ATP-7B and ATP-7A) have been found within the last 10 years. Aceruloplasminemia is characterized by mutations in the ceruloplasmin gene itself [8]. Aceruloplasminemia, after the first description, has been reported mainly in Japanese patients [1, 2, 9–18] and rarely in whites [10, 19]. More than 30 aceruloplasminemiacausing mutations have been identified. Some of the mutations have been found in several families throughout the world, while other have been detected in a single patient. The majority of mutations in the CP gene are the truncated mutations leading to the formation of a premature stop codon. These mutations would be predicted to result in formation of a protein lacking the copper-binding sites resumed to be critical for enzymatic function. First clinical manifestation is usually diabetes mellitus secondary to pancreatic involvement [2]. A progressive neurological disease begins a few years later, typically with focal dystonia, followed by dysarthria, extrapyramidal D. Di Raimondo A. Pinto A. Tuttolomondo P. Fernandez G. Licata Dipartimento Biomedico di Medicina Interna e Specialistica, AOU Policlinico ‘‘P. Giaccone’’, Universita degli Studi di Palermo, Palermo, Italy

Ethylene Receptor Antagonists: Strained Alkenes Are Necessary but Not Sufficient

Plants use ethylene as a hormone to control many physiological processes. Ethylene perception involves its binding to an unusual copper-containing, membrane-bound receptor. Inhibitors of ethylene action are valuable to study signaling and may have practical use in horticulture. Past investigation of alkene ligands for this receptor has identified strain as the key factor in antagonism of ethylene binding and action, consistent with known trends in metal-alkene complex stability. However, in this work, this principle could not be extended to other alkenes, prompting development of the proposal that a ring-opening reaction accounts for the unusual potency of cyclopropene ethylene antagonists. Another factor augmenting the affinity of alkenes for the copper binding site is pyramidalization, as in trans-cycloalkenes. The enantiomeric selectivity in the binding of one such alkene to the ethylene receptor demonstrates its protein-composed asymmetric environment.

Novel Role of Antioxidant-1 (Atox1) as a Copper-dependent Transcription Factor Involved in Cell Proliferation

Copper plays a fundamental role in regulating cell growth. Many types of human cancer tissues have higher copper levels than normal tissues. Copper can also induce gene expression. However, transcription factors that mediate copper-induced cell proliferation have not been identified in mammals. Here we show that antioxidant-1 (Atox1), previously appreciated as a copper chaperone, represents a novel copper-dependent transcription factor that mediates copper-induced cell proliferation. Stimulation of mouse embryonic fibroblasts (MEFs) with copper markedly increased cell proliferation, cyclin D1 expression, and entry into S phase, which were completely abolished in Atox1(-/-) MEFs. Promoter analysis and EMSA revealed that copper stimulates the Atox1 binding to a previously undescribed cis element in the cyclin D1 promoter. The ChIP assay confirms that copper stimulates Atox1 binding to the DNA in vivo. Transfection of Atox1 fused to the DNA-binding domain of Gal4 demonstrated a copper-dependent transactivation in various cell types, including endothelial and cancer cells. Furthermore, Atox1 translocated to the nucleus in response to copper through its highly conserved C-terminal KKTGK motif and N-terminal copper-binding sites. Finally, the functional role of nuclear Atox1 is demonstrated by the observation that re-expression of nuclear-targeted Atox1 in Atox1(-/-) MEFs rescued the defective copper-induced cell proliferation. Thus, Atox1 functions as a novel transcription factor that, when activated by copper, undergoes nuclear translocation, DNA binding, and transactivation, thereby contributing to cell proliferation.

Copper shares a piece of the π

Recent crystal structures of a bacterial copper tolerance protein reveal an intriguing copper binding site that includes tryptophan. Its close proximity coupled with spectroscopic data suggests an unusual cation-π interaction between Cu(I) and the aromatic ring of tryptophan.

Copper-induced structural changes in the ovine prion protein are influenced by a polymorphism at codon 112

Prion diseases are associated with conformational change in the copper-binding protein PrP. The copper-binding sites in PrP are located in the N-terminal region of the molecule and comprise a series of tandem repeats of the sequence PHGGGWGQ together with two histidines at residues 96 and 111 (human PrP numbering). The co-ordination of copper ions within the non-octapeptide repeat metal ion-binding site involves Met109 (human numbering, which corresponds with Met112 in ovine PrP) and the binding of copper to this site leads to an increase in β-sheet formation in PrP. Here we have investigated the influence of the M112T polymorphism on copper-induced structural changes in ovine recombinant PrP. M112ARQ and T112ARQ ovine PrP show similar secondary structure although M112ARQ appears more thermostable than T112ARQ. Following treatment with copper, M112ARQ showed a greater increase in β-sheet content than did T112ARQ when measured by CD spectroscopy and by ELISA using anti-PrP monoclonal antibodies. These biochemical and biophysical differences between M112ARQ and T112ARQ correlate with similar differences seen between allelic variants of ovine PrP associated with susceptibility and resistance to classical scrapie. These observations suggest that T112ARQ may provide a measure of resistance to classical scrapie pathogenesis compared to M112ARQ.

Delivery of the Cu-transporting ATPase ATP7B to the plasma membrane in Xenopus oocytes

Cu-transporting ATPase ATP7B (Wilson disease protein) is essential for the maintenance of intracellular copper concentration. In hepatocytes, ATP7B is required for copper excretion, which is thought to occur via a transient delivery of the ATP7B- and copper-containing vesicles to the apical membrane. The currently available experimental systems do not allow analysis of ATP7B at the cell surface. Using epitope insertion, we identified an extracellular loop into which the HA-epitope can be introduced without inhibiting ATP7B activity. The HA-tagged ATP7B was expressed in Xenopus oocytes and the presence of ATP7B at the plasma membrane was demonstrated by electron microscopy, freeze-fracture experiments, and surface luminescence measurements in intact cells. Neither the deletion of the entire N-terminal copper-binding domain nor the inactivating mutation of catalytic Asp1027 affected delivery to the plasma membrane of oocytes. In contrast, surface targeting was decreased for the ATP7B variants with mutations in the ATP-binding site or the intra-membrane copper-binding site, suggesting that ligand-stabilized conformation(s) are important for ATP7B trafficking. The developed system provides significant advantages for studies that require access to both sides of ATP7B in the membrane.

Copper binding sites in the C‐terminal domain of mouse prion protein: A hybrid (QM/MM) molecular dynamics study

We present a hybrid QM/MM Car-Parrinello molecular dynamics study of the copper-loaded C-terminal domain of the mouse prion protein. By means of a statistical analysis of copper coordination in known protein structures, we localized the protein regions with the highest propensity for copper ion binding. The identified candidate structures were subsequently refined via QM/MM simulations. Their EPR characteristics were computed to make contact with the experimental data and to probe the sensitivity to structural and chemical changes. Overall best agreement with the experimental EPR data (Van Doorslaer et al., J Phys Chem B 2001; 105: 1631-1639) and the information currently available in the literature is observed for a binding site involving H187. Moreover, a reinterpretation of the experimental proton hyperfine couplings was possible in the light of the present computational findings.

Molecular Dynamics Simulations of Two Tandem Octarepeats from the Mammalian Prion Protein: Fully Cu 2+-bound and Metal-Free Forms

Molecular dynamics simulations have been conducted on a model fragment (Ac-PHGGGWGQPHGGGW-NH 2) of the prion protein octarepeat domain, both in the Cu 2+-bound and metal-free forms. The copper-bound models are based on the consensus structure of the core Cu 2+-binding site of an individual octarepeat, relevant to the fully Cu 2+-occupied prion protein octarepeat region. The model peptides contain Cu 2+ bound through a His imidazole ring and two deprotonated amide N-atoms in the peptide backbone supplied by the following two Gly residues. Both the copper-bound and metal-free models have been simulated with the OPLS all-atom force field with the GROMACS molecular dynamics package. These simulations, with two tandem copper-binding sites, represent the minimum model necessary to observe potential structuring between the copper-binding sites in the octarepeat region. The GWGQ residues constitute a flexible linker region that predominantly adopts a turn, serving to bring adjacent His residues into close proximity. The consequent formation of stable structures demonstrates that the copper-bound octarepeat region allows the copper-coordinating sites to come into van der Waals contact, packing into particular orientations to further stabilize the bend in the GWGQ linker region.

A prophenoloxidase from the Chinese mitten crab Eriocheir sinensis: Gene cloning, expression and activity analysis

Prophenoloxidase (proPO) is a conserved copper-containing enzyme that plays important roles in immune response of crustaceans and insects. In the present study, the full-length cDNA of a prophenoloxidase (designated EsproPO) was cloned from haemocytes of Chinese mitten crab Eriocheir sinensis by expressed sequence tag (EST) and PCR techniques. The isolated 3549 bp full-length cDNA of EsproPO contained a 2040 bp open reading frame (ORF) encoding a putative proPO protein of 679 amino acids, a 5′-untranslated region (UTR) of 68 bp, and a long 3′-UTR of 1441 bp. Two putative copper-binding sites, a proteolytic activation site, and a complement-like motif (GCGWPQHM) were identified in the deduced amino acid sequence of EsproPO. Homology analysis revealed that EsproPO was highly similar to other proPOs from crustaceans with identities from 52% to 68%. The conserved domains and motifs, and higher similarity with other proPOs suggested that EsproPO was a member of the proPO family. The mRNA expression of EsproPO and PO specific activities in the tissues of hepatopancreas, gill, gonad, muscle, heart, eye and haemocytes were measured by quantitative real-time PCR and colorimetric assay, respectively. The mRNA transcripts of EsproPO and PO specific activities could be detected in all the examined tissues with the highest level both in hepatopancreas. Three peaks of EsproPO mRNA expression were recorded at 2 h, 12 h and 48 h in haemocytes of Chinese mitten crab post Vibrio anguillarum challenge, which was consistent with the temporal profile of PO specific activity. The mRNA expression pattern and the activity fluctuation of EsproPO post V. anguillarum stimulation indicated that it was potentially involved in the acute response against invading bacteria in Chinese mitten crab.

The axial ligand and extent of protein folding determine whether Zn or Cu binds to amicyanin

M98Q amicyanin is isolated with zinc bound to its type 1 copper-binding site. The influence of the axial ligand of the type 1 copper site on metal specificity is strongest prior to the completion of protein folding and adoption of the final type 1 site geometry. The preference for zinc over copper correlated with the selectivity of apoamicyanin in vitro in the partially folded, rather than the completely folded state. These results suggest that metal incorporation in vivo occurs during protein folding in the periplasm and not to a preformed type 1 site.

Optical Spectra of Cu(II)−Azurin by Hybrid TDDFT-Molecular Dynamics Simulations

The ground state electronic structure of oxidized azurin from Pseudomonas aeruginosa and its optical response have been investigated by combining hybrid quantum mechanics/molecular mechanics simulations with time-dependent density functional theory. In agreement with experiment, we find that the unpaired electron spin density is mainly localized on the copper ion. The vertical absorption spectrum in the visible range is well reproduced, with the central band centered around 2.1 eV. The anisotropic dipolar field due to the extended alpha-helix polarizes the metal binding site and is responsible for a shift of the absorption bands by +/-0.1-0.2 eV. At 300 K, the bond distances of the copper binding site undergo large fluctuations (approximately 0.3 A). It is crucial to take these thermal fluctuations into account for a faithful description of the optical properties.

Purification, Characterization, and Molecular Cloning of Tyrosinase fromPholiota nameko

Tyrosinase (monophenol, 3,4-dihydroxy L-phenylalanine (L-DOPA):oxygen oxidoreductase, EC 1.14.18.1) was isolated from fruit bodies of Pholiota nameko and purified to homogeneity. The purified enzyme was a monomer with a molecular weight of 42,000 and contained 1.9 copper atoms per molecule. The N-terminal of the purified enzyme could not be detected by Edman degradation, probably due to blocking, while the C-terminal sequence of the enzyme was determined to be -Ala-Ser-Val-Phe-OH. The amino acid sequence deduced by cDNA cloning was made up of 625 amino acid residues and contained two putative copper-binding sites highly conserved in tyrosinases from various organisms. The C-terminal sequence of the purified enzyme did not correspond to that of the deduced sequence, but agreed with Ala384-Ser385-Val386-Phe387 in sequence. When the encoded protein was truncated at Phe387, the molecular weight of the residual protein was calculated to be approximately 42,000. These results suggest that P. nameko tyrosinase is expressed as a proenzyme followed by specific cleavage to produce a mature enzyme.

A copper-binding site in the cytoplasmic domain of BACE1 identifies a possible link to metal homoeostasis and oxidative stress in Alzheimer's disease

The amyloidogenic processing pathway of the APP (amyloid precursor protein) generates Abeta (amyloid beta-peptide), the major constituent in Alzheimer's disease senile plaques. This processing is catalysed by two unusual membrane-localized aspartic proteinases, beta-secretase [BACE1 (beta-site APP-cleaving enzyme 1)] and the gamma-secretase complex. There is a clear link between APP processing and copper homoeostasis in the brain. APP binds copper and zinc in the extracellular domain and Abeta also binds copper, zinc and iron. We have found that a 24-residue peptide corresponding to the C-terminal domain of BACE1 binds a single copper(I) atom with high affinity through cysteine residues. We also observed that the cytoplasmic domain of BACE1 interacts with CCS, the dedicated copper chaperone for SOD1 (superoxide dismutase 1). Overproduction of BACE1 reduces SOD1 activity in cells. Consequently, SOD1 activity, cytosolic copper and ectodomain cleavage of APP are linked through BACE1.