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PubMed2006

Evidence for iron channeling in the Fet3p-Ftr1p high-affinity iron uptake complex in the yeast plasma membrane.

In high-affinity iron uptake in the yeast Saccharomyces cerevisiae, Fe(II) is oxidized to Fe(III) by the multicopper oxidase, Fet3p, and the Fe(III) produced is transported into the cell via the iron permease, Ftr1p. These two proteins are likely part of a heterodimeric or higher order complex in the yeast plasma membrane. We provide kinetic evidence that the Fet3p-produced Fe(III) is trafficked to Ftr1p for permeation by a classic metabolite channeling mechanism. We examine the (59)Fe uptake kinetics for a number of complexes containing mutant forms of both Fet3p and Ftr1p and demonstrate that a residue in one protein interacts with one in the other protein along the iron trafficking pathway as would be expected in a channeling process. We show that, as a result of some of these mutations, iron trafficking becomes sensitive to an added Fe(III) chelator that inhibits uptake in a strictly competitive manner. This inhibition is not strongly dependent on the chelator strength, however, suggesting that Fe(III) dissociation from the iron uptake complex, if it occurs, is kinetically slow relative to iron permeation. Metabolite channeling is a common feature of multifunctional enzymes. We constructed the analogous ferroxidase, permease chimera and demonstrate that it supports iron uptake with a kinetic pattern consistent with a channeling mechanism. By analogy to the Fe(III) trafficking that leads to the mineralization of the ferritin core, we propose that ferric iron channeling is a conserved feature of iron homeostasis in aerobic organisms.

Biological Transport↗
PubMed2006

Structural basis of the ferrous iron specificity of the yeast ferroxidase, Fet3p.

Fet3p is a multicopper oxidase (MCO) that functions together with the iron permease, Ftr1p, to support high-affinity Fe uptake in yeast. Fet3p is a ferroxidase that, like ceruloplasmin and hephaestin, couples the oxidation of 4 equiv of Fe(II) to the reduction of O2 to 2 H2O. The ferrous iron specificity of this subclass of MCO proteins has not been delineated by rigorous structure-function analysis. Here the crystal structure of Fet3p has been used as a template to identify the amino acid residues that confer this substrate specificity and then to quantify the contributions they make to this specific reactivity by thermodynamic and kinetic analyses. In terms of the Marcus theory of outer-sphere electron transfer, we show here that D283, E185, and D409 in Fet3p provide a Fe(II) binding site that actually favors ferric iron; this site thus reduces the reduction potential of the bound Fe(II) in comparison to that of aqueous ferrous iron, providing a thermodynamically more robust driving force for electron transfer. In addition, E185 and D409 constitute parts of the electron-transfer pathway from the bound Fe(II) to the protein's type 1 Cu(II). This electronic matrix coupling relies on H-bonds from the carboxylate OD2 atom of each residue to the NE2 NH group of the two histidine ligands at the type 1 Cu site. These two acidic residues and this H-bond network appear to distinguish a fungal ferroxidase from a fungal laccase since the specificity that Fet3p has for Fe(II) is completely lost in a Fet3pE185A/D409A mutant. Indeed, this double mutant functions kinetically better as a laccase, albeit a relatively inefficient one.

Amino Acid Substitution↗
PubMed1998

Spectroscopic and magnetic studies of human ceruloplasmin: identification of a redox-inactive reduced Type 1 copper site.

Ceruloplasmin is unique among the multicopper oxidases in that in addition to the usual copper stoichiometry of one Type 1 copper site and a Type 2/Type 3 trinuclear copper cluster, it contains two other Type 1 sites. This assignment of copper sites, based on copper quantitation, sequence alignment, and crystallography, is difficult to reconcile with the observed spectroscopy. Furthermore, some chemical or spectroscopic differences in ceruloplasmin have been reported depending on the method of purification. We have studied the resting (as isolated by a fast, one-step procedure) and peroxide-oxidized forms of human ceruloplasmin. Using a combination of X-ray absorption spectroscopy, a chemical assay, magnetic susceptibility, electron paramagnetic resonance spectroscopy, and absorption spectroscopy, we have determined that peroxide-oxidized ceruloplasmin contains one permanently reduced Type 1 site. This site is shown to have a reduction potential of approximately 1.0 V. Thus, one of the additional Type 1 sites in ceruloplasmin cannot be catalytically relevant in the form of the enzyme studied. Furthermore, the resting form of the enzyme contains an additional reducing equivalent, which is distributed among the remaining five copper sites as expected from their relative potentials. This may indicate that the resting form of ceruloplasmin in plasma under aerobic conditions is a four-electron oxidized form, which is consistent with its function in the four-electron reduction of dioxygen to water.

Binding Sites↗
PubMed1999

Investigation of the anomalous spectroscopic features of the copper sites in chicken ceruloplasmin: comparison to human ceruloplasmin.

Chicken ceruloplasmin has been previously reported to display a number of key differences relative to human ceruloplasmin: a lower copper content and a lack of a type 2 copper signal by electron paramagnetic resonance (EPR) spectroscopy. We have studied the copper sites of chicken ceruloplasmin in order to probe the origin of these differences, focusing on two forms of the enzyme: "resting" (as isolated by a fast, one-step procedure) and "peroxide-oxidized". From X-ray absorption, EPR, and UV/visible absorption spectroscopies, we have shown that all of the copper sites are oxidized in peroxide-oxidized chicken ceruloplasmin and that none of the type 1 copper sites display the EPR features typical for type 1 copper sites that lack an axial methionine. In the resting form, the type 2 copper center is reduced. Upon oxidation, it does not appear in the EPR spectrum at 77 K, but it can be observed by using magnetic susceptibility, EPR at approximately 8 K, and magnetic circular dichroism spectroscopy. It displays unusually fast relaxation, indicative of coupling with the adjacent type 3 copper pair of the trinuclear copper cluster. From reductive titrations, we have found that the reduction potential of the type 2 center is higher than those of the other copper sites, thus explaining why it is reduced in the resting form. These results provide new insight into the nature of the additional type 1 copper sites and the redox distribution among copper sites in the different ceruloplasmins relative to other multicopper oxidases.

Animals↗
PubMed2002

Toward synthetic analogues of linked redox and catalytic multimetal sites in proteins: a model of the histidine-cysteine bridged dicopper array.

Contiguous HisCys residues link a type 1 Cu electron-transfer site to a catalytic Cu-containing site in nitrite reductase and the multicopper oxidases. In efforts to understand the role of the linker in these multimetallic arrays and to design new catalysts, a mixed-valent dicopper complex comprising a bridging thiolate/N-donor ligand that models the CuHisCysCu motif was prepared and characterized by X-ray crystallography. Comparison of spectroscopic and cyclic voltammetry data to those of the mononuclear analogues of each portion of the complex, LCuSCPh(3) and LCu(py) (L = beta-diketiminate, py = pyridyl), confirmed retention of the dicopper structure in solution.

Binding Sites↗
PubMed2002

Nature of the intermediate formed in the reduction of O(2) to H(2)O at the trinuclear copper cluster active site in native laccase.

The multicopper oxidases contain at least four copper atoms and catalyze the four-electron reduction of O(2) to H(2)O at a trinuclear copper cluster. An intermediate, termed native intermediate, has been trapped by a rapid freeze-quench technique from Rhus vernicifera laccase when the fully reduced form reacts with dioxygen. This intermediate had been described as an oxygen-radical bound to the trinuclear copper cluster with one Cu site reduced. XAS, however, shows that all copper atoms are oxidized in this intermediate. A combination of EXAFS, multifrequency EPR, and VTVH MCD has been used to understand how this fully oxidized trinuclear Cu cluster relates to the fully oxidized resting form of the enzyme. It is determined that in the native intermediate all copper atoms of the cluster are bridged by the product of full O(2) reduction. In contrast, the resting form has one copper atom of the cluster (the T2 Cu) magnetically isolated from the others. The native intermediate decays to the resting oxidized form with a rate that is too slow to be in the catalytic cycle. Thus, the native intermediate appears to be the catalytically relevant fully oxidized form of the enzyme, and its role in catalysis is considered.

Binding Sites↗
PubMed2004

Aerial oxidation of primary alcohols and amines catalyzed by Cu(II) complexes of 2,2'-selenobis(4,6-di-tert-butylphenol) providing [O,Se,O]-donor atoms.

Seven copper(II)-complexes 1-7 with the ligand 2,2'-selenobis(4,6-di-tert-butylphenol) providing [O,Se,O]-donor atoms have been isolated and characterized. Three of them 1, 2 and 3 are mononuclear, two 4 and 7 dinuclear and 5, 6 are trinuclear. The crystal structures of the complexes were determined by X-ray diffraction and the electronic structures were established by various physical methods including EPR and variable temperature (2-290 K) susceptibility measurements. The magnetic behaviour of the compounds 4-6 exhibits antiferromagnetic exchange coupling resulting in well-isolated S(t)= 1/2 ground state for 5 and 6 and a diamagnetic spin state for 4. Complexes 5 and 6 belong to the class of asymmetric trinuclear copper(II) complexes modelling the trinuclear copper site in multicopper oxidases. Complex 1 is a catalyst in the presence of a strong base for the aerial oxidation of primary alcohols to the corresponding aldehydes. A dinuclear complex, seemed to be 4, prepared in situ has been found to be a catalyst for the aerial oxidation of primary amines containing alpha-C-H atoms. Primary kinetic isotope effects show that H-abstraction from the alpha-carbon atom of a coordinated substrate (alcoholato or amine) is the rate-determining step in both cases. Two functional models for the metalloenzymes galactose oxidase and amine oxidases are thus described.

Alcohols↗
PubMed2002

Characterization of a low redox potential laccase from the basidiomycete C30.

A new exocellular laccase was purified from the basidiomycete C30. LAC2 is an acidic protein (pI = 3.2) preferentially produced upon a combined induction by copper and p-hydroxybenzoate. The spectroscopic signature (UV/visible and EPR) of this isoform is typical of multicopper oxidases, but its enzymatic and physico-chemical properties proved to be markedly different from those of LAC1, the constitutive laccase previously purified from the same organism. In particular, the LAC2 kcat values observed for the oxidation of the substrates syringaldazine (kcat = 65 600 min-1), ABTS (2,2-azino-bis-[3-ethylthiazoline-6-sulfonate] (kcat = 41 000 min-1) and guaiacol (kcat = 75 680 min-1) are 10-40 times those obtained with LAC1 and the redox potential of its T1 copper is 0.17 V lower than that of LAC1 (E degrees = 0.73 V). This is the first report on a single organism producing simultaneously both a high and a low redox potential laccase. The cDNA, clac2, was cloned and sequenced. It encodes a protein of 528 amino acids that shares 69% identity (79% similarity) with LAC1 and 81% identity (95% similarity) with Lcc3-2 from Polyporus ciliatus (AF176321-1), its nearest neighbor in database. Possible reasons for why this basidiomycete produces, in vivo, enzyme forms with such different behaviors are discussed.

Azides↗
PubMed2003

A redox switch in CopC: an intriguing copper trafficking protein that binds copper(I) and copper(II) at different sites.

The protein CopC from Pseudomonas syringae has been found capable of binding copper(I) and copper(II) at two different sites, occupied either one at a time or simultaneously. The protein, consisting of 102 amino acids, is known to bind copper(II) in a position that is now found consistent with a coordination arrangement including His-1, Glu-27, Asp-89, and His-91. A full solution structure analysis is reported here for Cu(I)-CopC. The copper(I) site is constituted by His-48 and three of the four Met residues (40, 43, 46, 51), which are clustered in a Met-rich region. Both copper binding sites have been characterized through extended x-ray absorption fine structure studies. They represent novel coordination environments for copper in proteins. The two sites are approximately 30 A far apart and have little affinity for the ion in the other oxidation state. Oxidation of Cu(I)-CopC or reduction of Cu(II)-CopC causes migration of copper from one site to the other. This behavior is observed both in NMR and EXAFS studies and indicates that CopC can exchange copper between two sites activated by a redox switch. CopC resides in the periplasm of Gram-negative bacteria where there is a multicopper oxidase, CopA, which may modulate the redox state of copper. CopC and CopA are coded in the same operon, responsible for copper resistance. These peculiar and novel properties of CopC are discussed with respect to their relevance for copper homeostasis.

Amino Acid Sequence↗
PubMed1981

Internal duplication and evolution of human ceruloplasmin.

With the completion of the primary structure of the 50,000- and 19,000-dalton fragments of human ceruloplasmin [ferroxidase; iron(II):oxygen oxidoreductase, EC 1.16.3.1], over half of the covalent structure of the single polypeptide chain of this protein is known. Visual and computer analysis of the sequence of the 564 amino acid residues in the two fragments gives clear evidence of statistically significant internal homology suggestive of evolutionary replication of two smaller units. Two homology regions, each composed of 224 residues, were defined by an intrasequence alignment that required only three gaps in each 224-residue segment. The two homology regions exhibited 43% identity in sequence, and 13% of the remaining positions had similar residues. The sequence of a 160-residue segment in ceruloplasmin exhibits significant homology to the active (copper-binding) sites of blue electron-transfer proteins such as azurins and plastocyanins and multicopper oxidases such as cytochrome oxidase and superoxide dismutase. It is proposed that a primitive ceruloplasmin gene was formed by the fusion of two genes coding, respectively, for protein abut 160 and 190 amino acid residues in length and that this precursor gene coding for about 350 amino acids was later triplicated to form the gene for the present-day ceruloplasmin molecule of about 1050 amino acids.

Amino Acid Sequence↗
PubMed1984

Structural model of human ceruloplasmin based on internal triplication, hydrophilic/hydrophobic character, and secondary structure of domains.

A molecular model for the structure of human ceruloplasmin is proposed that is based on the determination of the complete amino acid sequence, studies of the products of limited proteolytic cleavage, calculations of the hydrophilic/hydrophobic character (hydropathy profile), and predictions of the local secondary structure. This multicopper oxidase (Mr approximately 132,000) consists of a single polypeptide chain (1046 amino acid residues) with four attached glucosamine oligosaccharides. Computer-assisted statistical analysis of the internal repetition in the amino acid sequence confirms that the entire polypeptide chain is divided into three contiguous homology units, each containing about 350 amino acid residues. Each homology unit is subdivided into three domains, designated A1, A2, and B, that differ in structure and probably in function. Calculations of the hydropathy profile and predictions of the secondary structure support a molecular model based on internal repetition of three homology units and help to identify characteristic features of the interdomain junctions. The alignment scores for internal duplication of pairings of the three homology units of ceruloplasmin exceed the scores yet reported for contiguous internal duplication of any other protein. This highly significant evidence for intragenic repetition suggests that the ceruloplasmin molecule evolved by tandem triplication of ancestral genes coding for a primordial copper oxidase.

Amino Acid Sequence↗
PubMed1997

Purification and characterization of Fet3 protein, a yeast homologue of ceruloplasmin.

The FET3 gene product of Saccharomyces cerevisiae is an essential component of the high affinity iron transport system. Based on FET3 sequence homology to the multicopper oxidase family and iron oxidation studies in spheroplasts (De Silva, D. M., Askwith, C. C., Eide, D., and Kaplan, J. (1995) J. Biol. Chem. 270, 1098-1101), it was hypothesized that the Fet3 protein (Fet3p) was a cell surface ferroxidase. To further characterize the protein, we have isolated Fet3p from yeast membranes and purified the protein to apparent homogeneity. Consistent with its localization at the plasma membrane, Fet3p is a glycosylated protein. SDS-polyacrylamide gel electrophoresis analysis showed that the protein was present in two differentially glycosylated forms of approximately 120 and 100 kDa. Purified Fet3p is a copper-containing protein that is able to catalyze the oxidation of a variety of organic compounds in addition to ferrous iron. Azide and metal chelators strongly inhibited enzyme activity. Iron appeared to be the best substrate for the enzyme, and the apparent Km for ferrous oxidation was 2 microM. Interestingly, Fet3p was able to effectively catalyze the incorporation of iron onto apotransferrin. We conclude that Fet3p is a ferro-O2-oxidoreductase in yeast, homologous to the human plasma protein ceruloplasmin.

Ceruloplasmin↗
PubMed1997

Biochemical characterization of the Wilson disease protein and functional expression in the yeast Saccharomyces cerevisiae.

Wilson disease is a disorder of copper metabolism characterized by hepatic cirrhosis and neuronal degeneration due to inherited mutations in a gene encoding a putative copper-transporting P-type ATPase. Polyclonal antisera generated against the amino terminus of the Wilson protein detected a specific 165-kDa protein in HepG2 and CaCo cell lysates. Further analysis revealed that this protein is synthesized as a single-chain polypeptide and localized to the trans-Golgi network under steady state conditions. An increase in the copper concentration resulted in the rapid movement of this protein to a cytoplasmic vesicular compartment. This copper-specific cellular redistribution of the Wilson protein is a reversible process that occurs independent of a new protein synthesis. Expression of the wild-type but not mutant Wilson protein in the ccc2Delta strain of Saccharomyces cerevisiae restored copper incorporation into the multicopper oxidase Fet3p, providing direct evidence of copper transport by the Wilson protein. Taken together these data reveal a remarkable evolutionary conservation in the cellular mechanisms of copper metabolism and provide a unique model for the regulation of copper transport into the secretory pathway of eucaryotic cells.

Adenosine Triphosphatases↗
PubMed1998

HAH1 is a copper-binding protein with distinct amino acid residues mediating copper homeostasis and antioxidant defense.

HAH1 is a 68-amino acid protein originally identified as a human homologue of Atx1p, a multi-copy suppressor of oxidative injury in sod1 delta yeast. Molecular modeling of HAH1 predicts a protein structure of two alpha-helices overlaying a four-stranded antiparallel beta-sheet with a potential metal binding site involving two conserved cysteine residues. Consistent with this model, in vitro studies with recombinant HAH1 directly demonstrated binding of Cu(I), and site-directed mutagenesis identified these cysteine residues as copper ligands. Expression of wild type and mutant HAH1 in atx1 delta yeast revealed the essential role of these cysteine residues in copper trafficking to the secretory compartment in vivo, as expression of a Cys-12/Cys-15 double mutant abrogated copper incorporation into the multicopper oxidase Fet3p. In contrast, mutation of the highly conserved lysine residues in the carboxyl terminus of HAH1 had no effect on copper trafficking to the secretory pathway but eliminated the antioxidant function of HAH1 in sod1 delta yeast. Taken together, these data support the concept of a unique copper coordination environment in HAH1 that permits this protein to function as an intracellular copper chaperone mediating distinct biological processes in eucaryotic cells.

Amino Acid Sequence↗
PubMed1998

Functional expression of the menkes disease protein reveals common biochemical mechanisms among the copper-transporting P-type ATPases.

Menkes disease is a fatal neurodegenerative disorder of childhood caused by the absence or dysfunction of a putative P-type ATPase encoded on the X chromosome. To elucidate the function of the Menkes disease protein, a plasmid containing the open reading frame of the human Menkes disease gene was constructed and used to transform a strain of Saccharomyces cerevisiae deficient in CCC2, the yeast Menkes/Wilson disease gene homologue. ccc2Delta yeast are deficient in copper transport into the secretory pathway, and expression of a wild type human Menkes cDNA complemented this defect, as evidenced by the restoration of copper incorporation into the multicopper oxidase Fet3p. Site-directed mutagenesis demonstrated the essential role of four specific amino acids in this process, including a conserved histidine, which is the site of the most common disease mutation in the homologous Wilson disease protein. The expression of Menkes cDNAs with successive mutations of the conserved cysteine residues in the six amino-terminal MXCXXC metal binding domains confirmed the essential role of these cysteine residues in copper transport but revealed that each of these domains is not functionally equivalent. These data demonstrate that the Menkes disease protein functions to deliver copper into the secretory pathway of the cell and that this process involves biochemical mechanisms common to previously characterized members of this P-type ATPase family.

Adenosine Triphosphatases↗
PubMed2002

Mechanisms of copper incorporation into human ceruloplasmin.

Ceruloplasmin is a multicopper oxidase essential for normal iron homeostasis. To elucidate the mechanisms of copper incorporation into this protein, holoceruloplasmin biosynthesis was examined by immunoblot analysis and (64)Cu metabolic labeling of Chinese hamster ovary cells transfected with cDNAs encoding wild-type or mutant ceruloplasmin. This analysis reveals that the incorporation of copper into newly synthesized apoceruloplasmin in vivo results in a detectable conformational change in the protein. Strikingly, despite the unique functional role of each copper site within ceruloplasmin, metabolic studies indicate that achieving this final conformation-driven state requires the occupation of all six copper-binding sites with no apparent hierarchy for copper incorporation at any given site. Consistent with these findings a missense mutation (G631R), resulting in aceruloplasminemia and predicted to alter the interactions at a single type I copper-binding site, results in the synthesis and secretion only of apoceruloplasmin. Analysis of copper incorporation into apoceruloplasmin in vitro reveals that this process is cooperative and that the failure of copper incorporation into copper-binding site mutants observed in vivo is intrinsic to the mutant proteins. These findings reveal a precise and sensitive mechanism for the formation of holoceruloplasmin under the limiting conditions of copper availability within the cell that may be generally applicable to the biosynthesis of cuproproteins within the secretory pathway.

Animals↗
PubMed2002

A key structural role for active site type 3 copper ions in human ceruloplasmin.

Human ceruloplasmin is a copper containing serum glycoprotein with multiple functions. The crystal structure shows that its six domains are arranged in three pairs with a pseudo-ternary axis. Both the holo and apo forms of human ceruloplasmin were studied by size exclusion chromatography and small angle x-ray scattering in solution. The experimental curve of the holo form displays conspicuous differences with the scattering pattern calculated from the crystal structure. Once the carbohydrate chains and flexible loops not visible in the crystal are accounted for, remaining discrepancies suggest that the central pair of domains may move as a whole with respect to the rest of the molecule. The quasisymmetrical crystal structure therefore appears to be stabilized by crystal packing forces. Upon copper removal, the scattering pattern of human ceruloplasmin exhibits very large differences with that of the holoprotein, which are interpreted in terms of essentially preserved domains freely moving in solution around flexible linkers and exploring an ensemble of open conformations. This model, which is supported by the analysis of domain interfaces, provides a structural explanation for the differences in copper reincorporation into the apoprotein and activity recovery between human ceruloplasmin and two other multicopper oxidases, ascorbate oxidase and laccase. Our results demonstrate that, beyond catalytic activity, the three-copper cluster at the N-terminal-C-terminal interface plays a crucial role in the structural stability of human ceruloplasmin.

Binding Sites↗