Copper transport and its defect in Wilson disease: characterization of the copper-binding domain of Wilson disease ATPase

Copper is an essential trace element which forms an integral component of many enzymes. While trace amounts of copper are needed to sustain life, excess copper is extremely toxic. An attempt is made here to present the current understanding of the normal transport of copper in relation to the absorption, intracellular transport and toxicity. Wilson disease is a genetic disorder of copper transport resulting in the accumulation of copper in organs such as liver and brain which leads to progressive hepatic and neurological damage. The gene responsible for Wilson disease (ATP7B) is predicted to encode a putative copper-transporting P-type ATPase. An important feature of this ATPase is the presence of a large N-terminal domain that contains six repeats of a copper-binding motif which is thought to be responsible for binding this metal prior to its transport across the membrane. We have cloned, expressed and purified the N-terminal domain (approximately 70 kD) of Wilson disease ATPase. Metal-binding properties of the domain showed the protein to bind several metals besides copper; however, copper has a higher affinity for the domain. The copper is bound to the domain in Cu(I) form with a copper: protein ratio of 6.5:1. X-ray absorption studies strongly suggest Cu(I) atoms are ligated to cysteine residues. Circular dichroism spectral analyses suggest both secondary and tertiary structural changes upon copper binding to the domain. Copper-binding studies suggest some degree of cooperativity in binding of copper. These studies as well as detailed structural information of the copper-binding domain will be crucial in determining the specific role played by the copper-transporting ATPase in the homeostatic control of copper in the body and how the transport of copper is interrupted by mutations in the ATPase gene.     

Identification of a vacuole-associated metalloreductase and its role in Ctr2-mediated intracellular copper mobilization

Copper is an essential trace metal whose biological utility is derived from its ability to cycle between oxidized Cu(II) and reduced Cu(I). Ctr1 is a high affinity plasma membrane copper permease, conserved from yeast to humans, that mediates the physiological uptake of Cu(I) from the extracellular environment. In the baker's yeast Saccharomyces cerevisiae, extracellular Cu(II) is reduced to Cu(I) via the action of the cell surface metalloreductase Fre1, similar to the human gp91(phox) subunit of the NADPH oxidase complex, which utilizes heme and flavins to catalyze electron transfer. The S. cerevisiae Ctr2 protein is structurally similar to Ctr1, localizes to the vacuole membrane, and mobilizes vacuolar copper stores to the cytosol via a mechanism that is not well understood. Here we show that Ctr2-1, a mutant form of Ctr2 that mislocalizes to the plasma membrane, requires the Fre1 plasma membrane metalloreductase for Cu(I) import. The conserved methionine residues that are essential for Ctr1 function at the plasma membrane are also essential for Ctr2-1-mediated Cu(I) uptake. We demonstrate that Fre6, a member of the yeast Fre1 metalloreductase protein family, resides on the vacuole membrane and functions in Ctr2-mediated vacuolar copper export, and cells lacking Fre6 phenocopy the Cu-deficient growth defect of ctr2Delta cells. Furthermore, both CTR2 and FRE6 mRNA levels are regulated by iron availability. Taken together these studies suggest that copper movement across intracellular membranes is mechanistically similar to that at the plasma membrane. This work provides a model for communication between the extracellular Cu(I) uptake and the intracellular Cu(I) mobilization machinery.     

characterization of the cytochrome c oxidase assembly factor Cox19 of Saccharomyces cerevisiae

Cox19 is an important accessory protein in the assembly of cytochrome c oxidase in yeast. The protein is functional when tethered to the mitochondrial inner membrane, suggesting its functional role within the intermembrane space. Cox19 resembles Cox17 in having a twin CX(9)C sequence motif that adopts a helical hairpin in Cox17. The function of Cox17 appears to be a Cu(I) donor protein in the assembly of the copper centers in cytochrome c oxidase. Cox19 also resembles Cox17 in its ability to coordinate Cu(I). Recombinant Cox19 binds 1 mol eq of Cu(I) per monomer and exists as a dimeric protein. Cox19 isolated from the mitochondrial intermembrane space contains variable quantities of copper, suggesting that Cu(I) binding may be a transient property. Cysteinyl residues important for Cu(I) binding are also shown to be important for the in vivo function of Cox19. Thus, a correlation exists in the ability to bind Cu(I) and in vivo function.     

Structural studies of the Alzheimer's amyloid precursor protein copper-binding domain reveal how it binds copper ions

Alzheimer's disease (AD) is the major cause of dementia. Amyloid beta peptide (Abeta), generated by proteolytic cleavage of the amyloid precursor protein (APP), is central to AD pathogenesis. APP can function as a metalloprotein and modulate copper (Cu) transport, presumably via its extracellular Cu-binding domain (CuBD). Cu binding to the CuBD reduces Abeta levels, suggesting that a Cu mimetic may have therapeutic potential. We describe here the atomic structures of apo CuBD from three crystal forms and found they have identical Cu-binding sites despite the different crystal lattices. The structure of Cu(2+)-bound CuBD reveals that the metal ligands are His147, His151, Tyr168 and two water molecules, which are arranged in a square pyramidal geometry. The site resembles a Type 2 non-blue Cu center and is supported by electron paramagnetic resonance and extended X-ray absorption fine structure studies. A previous study suggested that Met170 might be a ligand but we suggest that this residue plays a critical role as an electron donor in CuBDs ability to reduce Cu ions. The structure of Cu(+)-bound CuBD is almost identical to the Cu(2+)-bound structure except for the loss of one of the water ligands. The geometry of the site is unfavorable for Cu(+), thus providing a mechanism by which CuBD could readily transfer Cu ions to other proteins.     

A critical overview of the chemistry of copper-dependent low density lipoprotein oxidation: roles of lipid hydroperoxides, alpha-tocopherol, thiols, and ceruloplasmin

The mechanisms by which low-density lipoprotein (LDL) particles undergo oxidative modification to an atherogenic form that is taken up by the macrophage scavenger-receptor pathway have been the subject of extensive research for almost two decades. The most common method for the initiation of LDL oxidation in vitro involves incubation with Cu(II) ions. Although various mechanisms have been proposed to explain the ability of Cu(II) to promote LDL modification, the precise reactions involved in initiating the process remain a matter of contention in the literature. This review provides a critical overview and evaluation of the current theories describing the interactions of copper with the LDL particle. Following discussion of the thermodynamics of reactions dependent upon the decomposition of preexisting lipid hydroperoxides, which are present in all crude LDL preparations, attention is turned to the more difficult (but perhaps more physiologically-relevant) system of the hydroperoxide-free LDL particle. In both systems, the key role of alpha-tocopherol is discussed. In addition to its protective, radical-scavenging action, alpha-tocopherol can also behave as a prooxidant via its reduction of Cu(II) to Cu(I). Generation of Cu(I) greatly facilitates the decomposition of lipid hydroperoxides to chain-carrying radicals, but the mechanisms by which the vitamin promotes LDL oxidation in the absence of preformed hydroperoxides remain more speculative. In addition to the so-called tocopherol-mediated peroxidation model, in which polyunsaturated fatty acid oxidation is initiated by the alpha-tocopheroxyl radical (generated during the reduction of Cu(II) by alpha-tocopherol), an evaluation of the role of the hydroxyl radical is provided. Important interactions between copper ions and thiols are also discussed, particularly in the context of cell-mediated LDL oxidation. Finally, the mechanisms by which ceruloplasmin, a copper-containing plasma protein, can bring about LDL modification are discussed. Improved understanding of the mechanisms of LDL oxidation by copper ions should facilitate the establishment of any physiological role of the metal in LDL modification. It will also assist in the interpretation of studies in which copper systems of LDL oxidation are used in vitro to evaluate potential antioxidants.     

Cu(I) affinities of the domain 1 and 3 sites in the human metallochaperone for Cu,Zn-superoxide dismutase

The delivery of copper by the human metallochaperone CCS is a key step in the activation of Cu,Zn-superoxide dismutase (SOD1). CCS is a three-domain protein with Cu(I)-binding CXXC and CXC motifs in domains 1 and 3, respectively. A detailed analysis of the binding of copper to CCS, including variants in which the Cys residues from domains 1 and 3 have been mutated to Ser, and also using separate domain 1 and 3 constructs, demonstrates that CCS is able to bind 1 equiv of Cu(I) in both of these domains. The Cu(I) affinity of domain 1 is approximately 5 × 10(17) M(-1) at pH 7.5, while that of domain 3 is at least 1 order of magnitude weaker. The CXXC site will therefore be preferentially loaded with Cu(I), suggesting that domain 1 plays a role in the acquisition of the metal. The delivery of copper to the target occurs via domain 3 whose structural flexibility and ability to be transiently metalated during copper delivery appear to be more important than the Cu(I) affinity of its CXC motif. The Cu(I) affinity of domain 1 of CCS is comparable to that of HAH1, another cytosolic copper metallochaperone. CCS and HAH1 readily exchange Cu(I), providing a mechanism whereby cross-talk can occur between copper trafficking pathways.     

X-ray absorption spectroscopy of the copper chaperone HAH1 reveals a linear two-coordinate Cu(I) center capable of adduct formation with exogenous thiols and phosphines

The human copper chaperone HAH1 transports copper to the Menkes and Wilson proteins, which are copper-translocating P-type ATPases located in the trans-Golgi apparatus and believed to provide copper for important enzymes such as ceruloplasmin, tyrosinase, and peptidylglycine monooxygenase. Although a substantial amount of structural data exist for HAH1 and its yeast and bacterial homologues, details of the copper coordination remain unclear and suggest the presence of two protein-derived cysteine ligands and a third exogenous thiol ligand. Here we report the preparation and reconstitution of HAH1 with Cu(I) using a protocol that minimizes the use of thiol reagents believed to be the source of the third ligand. We show by x-ray absorption spectroscopy that this reconstitution protocol generates an occupied Cu(I) binding site with linear biscysteinate coordination geometry, as evidenced by (i) an intense edge absorption centered at 8982.5 eV, with energy and intensity identical to the rigorously linear two-coordinate model complex bis-2,3,5,6-tetramethylbenzene thiolate Cu(I) and (ii) an EXAFS spectrum that could be fit to two Cu-S interactions at 2.16 A, a distance typical of digonal Cu(I) coordination. Binding of exogenous ligands (GSH, dithiothreitol, and tris-(2-carboxyethyl)-phosphine) to the Cu(I) was investigated. When GSH or dithiothreitol was added to the chaperone during the reconstitution procedure, the resulting Cu(I)- HAH1 remained two-coordinate, whereas the addition of the phosphine during reconstitution elicited a three-coordinate species. When the exogenous ligands were titrated into the Cu(I)-HAH1, all formed three-coordinate adducts but with differing affinities. Thus, GSH and dithiothreitol showed weaker binding, with estimated KD values in the range 10-25 mm, whereas tris-(2-carboxyethyl)-phosphine showed stronger affinity, with a KD value of <5 mm. The implications of these findings for mechanisms of copper transport are discussed.     

Effects of copper on mammalian cell components

Both deficiency and excess of copper induce toxic effects on mammalian cell systems in vivo and in vitro. The effects can be related to the affinities of Cu(II) ions for specific cell components. The nucleus is a potential site for temporary Cu storage while primary targets for free Cu(II) ions are the thiol groups which reduce the ions to Cu(I). Cu(II) ions show a high affinity for nucleic acids, binding with DNA both at intrastrand and interstrand levels, possibly through intercalation between GC pairs. The ability to chelate Cu(II) ions is seen to be of the order: purine greater than purine ribonucleotides greater than purine ribonucleoside greater than pyrimidine ribonucleotides. Copper is an integral part of enzyme activation and enters into the molecular structure of several proteins, like ceruloplasmin. Cu(II) ion is a potential mutagenic agent as seen by its property of inducing infidelity in DNA synthesis in vitro. Teratogenic activities of copper have been reported but carcinogenicity is not yet confirmed. Copper is an essential component of chromatin and is known to accumulate preferentially in the heterochromatic regions. External application of higher doses, however, induces both clastogenic effects and spindle disturbances. In certain forms, inorganic copper enhances the clastogenic activity of other agents. The most widely studied human genetic maladies linked with copper metabolism are Menkes' and Wilson's diseases. Several mutations are known which influence Cu homeostasis in mammals. Such mutations in mice have been used extensively for biochemical studies.     

Role of N-terminal methionine residues in the redox activity of copper bound to alpha-synuclein

Amyloid aggregation of α-synuclein (AS) is one of the hallmarks of Parkinson’s disease. The interaction of copper ions with the N-terminal region of AS promotes its amyloid aggregation and metal-catalyzed oxidation has been proposed as a plausible mechanism. The AS(1–6) fragment represents the minimal sequence that models copper coordination to this intrinsically disordered protein. In this study, we evaluated the role of methionine residues Met1 and Met5 in Cu(II) coordination to the AS(1–6) fragment, and in the redox activity of the Cu–AS(1–6) complex. Spectroscopic and electronic structure calculations show that Met1 may play a role as an axial ligand in the Cu(II)–AS(1–6) complex, while Met5 does not participate in metal coordination. Cyclic voltammetry and reactivity studies demonstrate that Met residues play an important role in the reduction and reoxidation processes of this complex. However, Met1 plays a more important role than Met5, as substitution of Met1 by Ile decreases the reduction potential of the Cu–AS(1–6) complex by ~80 mV, causing a significant decrease in its rate of reduction. Reoxidation of the complex by oxygen results in oxidation of the Met residues to sulfoxide, being Met1 more susceptible to copper-catalyzed oxidation than Met5. The sulfoxide species can suffer elimination of methanesulfenic acid, rendering a peptide with no thioether moiety, which would impair the ability of AS to bind Cu(I) ions. Overall, our study underscores the important roles that Met1 plays in copper coordination and the reactivity of the Cu–AS complex.     

The lumenal loop Met672-Pro707 of copper-transporting ATPase ATP7A binds metals and facilitates copper release from the intramembrane sites

The copper-transporting ATPase ATP7A has an essential role in human physiology. ATP7A transfers the copper cofactor to metalloenzymes within the secretory pathway; inactivation of ATP7A results in an untreatable neurodegenerative disorder, Menkes disease. Presently, the mechanism of ATP7A-mediated copper release into the secretory pathway is not understood. We demonstrate that the characteristic His/Met-rich segment Met(672)-Pro(707) (HM-loop) that connects the first two transmembrane segments of ATP7A is important for copper release. Mutations within this loop do not prevent the ability of ATP7A to form a phosphorylated intermediate during ATP hydrolysis but inhibit subsequent dephosphorylation, a step associated with copper release. The HM-loop inserted into a scaffold protein forms two structurally distinct binding sites and coordinates copper in a mixed His-Met environment with an ～2:1 stoichiometry. Binding of either copper or silver, a Cu(I) analog, induces structural changes in the loop. Mutations of 4 Met residues to Ile or two His-His pairs to Ala-Gly decrease affinity for copper. Altogether, the data suggest a two-step process, where copper released from the transport sites binds to the first His(Met)(2) site, triggering a structural change and binding to a second 2-coordinate His-His or His-Met site. We also show that copper binding within the HM-loop stabilizes Cu(I) and protects it from oxidation, which may further aid the transfer of copper from ATP7A to acceptor proteins. The mechanism of copper entry into the secretory pathway is discussed.     

Copper binding traps the folded state of the SCO protein from Bacillus subtilis

The SCO protein from the aerobic bacterium Bacillus subtilis (BsSCO) is involved in the assembly of the cytochrome c oxidase complex, and specifically with the Cu(A) center. BsSCO has been proposed to play various roles in Cu(A) assembly including, the direct delivery of copper ions to the Cu(A) site, and/or maintaining the appropriate redox state of the cysteine ligands during formation of Cu(A). BsSCO binds copper in both Cu(II) and Cu(I) redox states, but has a million-fold higher affinity for Cu(II). As a prerequisite to kinetic studies, we measured equilibrium stability of oxidized, reduced and Cu(II)-bound BsSCO by chemical and thermal induced denaturation. Oxidized and reduced apo-BsSCO exhibit two-state behavior in both chemical- and thermal-induced unfolding. However, the Cu(II) complex of BsSCO is stable in up to nine molar urea. Thermal or guanidinium-induced unfolding of BsSCO-Cu(II) ensues only as the Cu(II) species is lost. The effect of copper (II) on the folding of BsSCO is complicated by a rapid redox reaction between copper and reduced, denatured BsSCO. When denatured apo-BsSCO is refolded in the presence of copper (II) some of the population is recovered as the BsSCO-Cu(II) complex and some is oxidized indicating that refolding and oxidation are competing processes. The proposed functional roles for BsSCO in vivo require that its cysteine residues are reduced and the presence of copper during folding may be detrimental to BsSCO attaining its functional state.     

A solution study of the interaction of the Cu(II) ions with HisGlyGlyTrp tetrapeptide and its evaluation as superoxide dismutase mimetic complex

The superoxide anion radical is a highly reactive toxic species produced during the metabolic processes. A number of copper (II) complexes with amino acids and peptides are known to show superoxide dismutase (SOD) like activity. The design and application of synthetic low molecular weight metal complexes as SOD mimics have received considerable attention during the last decade. A variety of di- and tri-peptides containing histidyl residue in different positions have been employed to bind Cu(II) and to show the activity. But reports on Cu(II) complex with tetra-peptide having histidine amino acid in this regard are limited. As the HGGGW peptide having His at its N-terminal is reported to be a potential moiety for Cu(2+) binding, in the present work the synthesis of HisGlyGlyTrp peptide and its complexation with copper (II) ions has been reported. The interaction of synthesized peptide with Cu(II) was studied by electron spray ionization-mass spectrometer (ESI-MS) and UV-Vis spectroscopic methods. The species distribution was studied by combined spectrophotometric and potentiometric methods. The studies were performed at 25 ± 0.1 °C with constant ionic strength (μ = 0.1 M NaNO(3)) in aqueous solution using Bjerrum-Calvin's pH-titration technique as adopted by Irving and Rossotti for binary systems. The solution studies suggested that the pH of the medium play important role in the different species formation of the copper complexes. Species distribution curves indicate that Cu complexation takes place at all physiological pH values from 3-11. The resultant copper (II) peptide complex at physiological pH was tested for superoxide dismutase activity using standard NBT method. The complex has SOD activity with the IC(50) value of 1.32 μM.     

A thiol oxidation interpretation of the Cu2+ effects on rat liver mitochondria

A comparative study of the Cu2+ effects, binding and reduction, has been performed on rat liver mitochondria. In the first minutes, Cu2+ (less than or equal to 50 micron) is massively bound and reduced to the extent of 70%-80% while a simultaneous activation of respiration takes place. Then the remaining 20% or so of Cu2+ are progressively bound and reduced while respiratory inhibition, Ca2+ and Mg2+ effluxes, and swelling are observed. EDTA, used as a copper chelator, prevents or reduces the copper effects and removes part of the bound copper, according to the time of introduction in the incubation medium after Cu2+. The results suggest that the two steps of the copper binding and the effects following involve mainly first the outer (cytosol side) proteins of the inner membrane and then those of the inner membrane. 100 microM dithiothreitol and 100 microM glutathione used as antioxidant thiol reagents prevent, as does EDTA, but do not reverse the 25 microM copper effects. They also decrease the copper binding; however, no relationship between binding and preventive action is observed. It is shown that glutathione and dithiothreitol have a specific potent ability to reduce Cu2+, which explains that in presence of these reagents copper may react with mitochondria partly or entirely in the form of Cu+. These findings suggest that Cu2+ in its Cu+ form has no mitochondrial effect. A mechanism of copper action involving oxidation of some membrane thiol groups is discussed.     

Copper binding to the amyloid-beta (Abeta) peptide associated with Alzheimer's disease: folding, coordination geometry, pH dependence, stoichiometry, and affinity of Abeta-(1-28): insights from a range of complementary spectroscopic techniques

There is now direct evidence that copper is bound to amyloid-beta peptide (Abeta) in senile plaque of Alzheimer's disease. Copper is also linked with the neurotoxicity of Abeta and free radical damage, and Cu(2+) chelators represent a possible therapy for Alzheimer's disease. We have therefore used a range of complementary spectroscopies to characterize the coordination of Cu(2+) to Abeta in solution. The mode of copper binding is highly pH-dependent. EPR spectroscopy indicates that both coppers have axial, Type II coordination geometry, square-planar or square-pyramidal, with nitrogen and oxygen ligands. Circular dichroism studies indicate that copper chelation causes a structural transition of Abeta. Competition studies with glycine and l-histidine indicate that copper binds to Abeta-(1-28) at pH 7.4 with an affinity of K(a) approximately 10(7) m(-1). (1)H NMR indicates that histidine residues are involved in Cu(2+) coordination but that Tyr(10) is not. Studies using analogues of Abeta-(1-28) in which each of the histidine residues have been replaced by alanine or in which the N terminus is acetylated suggest that the N terminus and His(13) are crucial for Cu(2+) binding and that His(6) and His(14) are also implicated. Evidence for the link between Alzheimer's disease and Cu(2+) is growing, and our studies have made a significant contribution to understanding the mode of Cu(2+) binding to Abeta in solution.     

Identification of hepatic copper-binding proteins from tilapia by column chromatography with proteomic approaches

Although copper is an essential element, it shows cytotoxic effects when present in excessive amounts with the production of hydroxyl radicals, which can damage phospholipids and enzymes. This necessitates a tight cellular control mechanism for copper homeostasis including its uptake and removal. The high copper contents in the liver of tilapia make this fish a suitable model for the study of copper binding proteins (CBPs). The liver was dissected from tilapia injected with Cu(2+) and cytosolic fractions were separated by using Superdex 75 column chromatography followed by atomic absorption spectrometry. Fractions in two major peaks containing CBPs were analyzed by using differential proteomic approaches, and loaded on a Cu chelating ion-immobilized affinity column (Cu-IMAC). Of the 113 differentially expressed proteins in these two peaks, 28 proteins were found to have copper binding ability, including well-characterized CBPs, such as copper transporter ATP7A and metallothionein. The networks of CBPs built up by Ingenuity Pathway Analysis (IPA) would help us to understand the transportation pathway and function of CBPs, which were related to free radical scavenging, cellular development and lipid metabolism. In addition, our results suggest that Cu(2+) would compete with Fe(2+) and Ca(2+) in binding with some target proteins, such as ferritin, transferrin, and calmodulin.     

The Methylococcus capsulatus (Bath) Secreted Protein, MopE*, Binds Both Reduced and Oxidized Copper

Under copper limiting growth conditions the methanotrophic bacterium Methylococcus capsulatus (Bath) secrets essentially only one protein, MopE*, to the medium. MopE* is a copper-binding protein whose structure has been determined by X-ray crystallography. The structure of MopE* revealed a unique high affinity copper binding site consisting of two histidine imidazoles and one kynurenine, the latter an oxidation product of Trp130. In this study, we demonstrate that the copper ion coordinated by this strong binding site is in the Cu(I) state when MopE* is isolated from the growth medium of M. capsulatus. The conclusion is based on X-ray Near Edge Absorption spectroscopy (XANES), and Electron Paramagnetic Resonance (EPR) studies. EPR analyses demonstrated that MopE*, in addition to the strong copper-binding site, also binds Cu(II) at two weaker binding sites. Both Cu(II) binding sites have properties typical of non-blue type II Cu (II) centres, and the strongest of the two Cu(II) sites is characterised by a relative high hyperfine coupling of copper (A_|| = 20 mT). Immobilized metal affinity chromatography binding studies suggests that residues in the N-terminal part of MopE* are involved in forming binding site(s) for Cu(II) ions. Our results support the hypothesis that MopE plays an important role in copper uptake, possibly making use of both its high (Cu(I) and low Cu(II) affinity properties.     

Acquisition of ionic copper by the bacterial outer membrane protein OprC through a novel binding site

Copper, while toxic in excess, is an essential micronutrient in all kingdoms of life due to its essential role in the structure and function of many proteins. Proteins mediating ionic copper import have been characterised in detail for eukaryotes, but much less so for prokaryotes. In particular, it is still unclear whether and how gram-negative bacteria acquire ionic copper. Here, we show that Pseudomonas aeruginosa OprC is an outer membrane, TonB-dependent transporter that is conserved in many Proteobacteria and which mediates acquisition of both reduced and oxidised ionic copper via an unprecedented CxxxM-HxM metal binding site. Crystal structures of wild-type and mutant OprC variants with silver and copper suggest that acquisition of Cu(I) occurs via a surface-exposed “methionine track” leading towards the principal metal binding site. Together with whole-cell copper quantitation and quantitative proteomics in a murine lung infection model, our data identify OprC as an abundant component of bacterial copper biology that may enable copper acquisition under a wide range of conditions.

How do Gram-negative bacteria acquire copper? This study shows that the outer membrane protein OprC from Pseudomonas aeruginosa is abundant during infection and mediates highly selective acquisition of both copper redox states via an extracellular "methionine track" and an unprecedented near-irreversible binding site.     

Mechanism of Cu,Zn-superoxide dismutase activation by the human metallochaperone hCCS

The mechanism for copper loading of the antioxidant enzyme copper, zinc superoxide dismutase (SOD1) by its partner metallochaperone protein is not well understood. Here we show the human copper chaperone for Cu,Zn-SOD1 (hCCS) activates either human or yeast enzymes in vitro by direct protein to protein transfer of the copper cofactor. Interestingly, when denatured with organic solvents, the apo-form of human SOD1 cannot be reactivated by added copper ion alone, suggesting an additional function of hCCS such as facilitation of an active folded state of the enzyme. While hCCS can bind several copper ions, metal binding studies in the presence of excess copper scavengers that mimic the intracellular chelation capacity indicate a limiting stoichiometry of one copper and one zinc per hCCS monomer. This protein is active and unlike the yeast protein, is a homodimer regardless of copper occupancy. Matrix-assisted laser desorption ionization-mass spectrometry and metal binding studies suggest that Cu(I) is bound by residues from the first and third domains and no bound copper is detected for the second domain of hCCS in either the full-length or truncated forms of the protein. Copper-induced conformational changes in the essential C-terminal peptide of hCCS are consistent with a "pivot, insert, and release" mechanism that is similar to one proposed for the well characterized metal handling enzyme, mercuric ion reductase.     

Interactions of flavonoids with iron and copper ions: a mechanism for their antioxidant activity

The metal chelating properties of flavonoids suggest that they may play a role in metal-overload diseases and in all oxidative stress conditions involving a transition metal ion. A detailed study has been made of the ability of flavonoids to chelate iron (including Fe 3+ ) and copper ions and its dependence of structure and pH. The acid medium may be important in some pathological conditions. In addition, the ability of flavonoids to reduce iron and copper ions and their activity-structure relationships were also investigated. To fulfil these objectives, flavones (apigenin, luteolin, kaempferol, quercetin, myricetin and rutin), isoflavones (daidzein and genistein), flavanones (taxifolin, naringenin and naringin) and a flavanol (catechin) were investigated. All flavonoids studied show higher reducing capacity for copper ions than for iron ions. The flavonoids with better Fe 3+ reducing activity are those with a 2,3-double bond and possessing both the catechol group in the B-ring and the 3-hydroxyl group. The copper reducing activity seems to depend largely on the number of hydroxyl groups. The chelation studies were carried out by means of ultraviolet spectroscopy and electrospray ionisation mass spectrometry. Only flavones and the flavanol catechin interact with metal ions. At pH 7.4 and pH 5.5 all flavones studied appear to chelate Cu 2+ at the same site, probably between the 5-hydroxyl and the 4-oxo groups. Myricetin and quercetin, however, at pH 7.4, appear to chelate Cu 2+ additionally at the ortho -catechol group, the chelating site for catechin with Cu 2+ at pH 7.4. Chelation studies of Fe 3+ to flavonoids were investigated only at pH 5.5. Only myricetin and quercetin interact strongly with Fe 3+ , complexation probably occurring again between the 5-hydroxyl and the 4-oxo groups. Their behaviour can be explained by their ability to reduce Fe 3+ at pH 5.5, suggesting that flavonoids reduce Fe 3+ to Fe 2+ before association.