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.     

Chicken ceruloplasmin. Evidence in support of a trinuclear cluster involving type 2 and 3 copper centers

Ceruloplasmin was isolated to purity from chicken plasma by a single-step chromatography on amino-ethyl-derivatized Sepharose. Molecular mass, as estimated by nonreducing sodium dodecyl sulfate-electrophoresis, was approximately 140 kDa, slightly higher than that found for ceruloplasmins from other sources. Specific activity as p-phenylenediamine oxidase was five times higher than that reported for mammalian ceruloplasmins. The copper content was estimated to be 5.01 +/- 0.35 atoms per protein molecule, 50% of which was EPR-detectable. The EPR spectrum was completely devoid of any signal typical of the type 2 copper as seen in the other blue multicopper oxidases and in ceruloplasmin from mammalian species. Anaerobic reduction of chicken ceruloplasmin resulted in the disappearance of the 330 nm optical band typical of type 3 copper, which was followed by the appearance of an EPR signal typical of type 2 copper. Subsequently, the type 1 copper and finally the newly formed type 2 copper were reduced. The original optical and EPR spectra were recovered within few minutes upon exposure of reduced ceruloplasmin to air. It is concluded that in oxidized chicken ceruloplasmin type 2 copper interacts with the diamagnetic pair responsible for the 330 nm absorption in such a way as to become EPR-undetectable and that the interaction is relieved by reduction of the pair. Whether this interaction is intrinsically weaker in other blue oxidases and ceruloplasmins studied or is lost with standard preparation procedures remains to be established.     

Interaction of nitric oxide with ceruloplasmin lacking an EPR-detectable type 2 copper.

Nitric oxide (NO) has previously been reported to modify the EPR spectrum of multicopper blue oxidases, disclosing a pure type 2 copper and inducing half-field transitions at g = 4. In the present work the reactivity of NO was reinvestigated with respect to ceruloplasmins having an apparently EPR-silent type 2 copper in their native state. The optical properties of NO-treated ceruloplasmin were independent of the initial redox state of the metal sites. Addition of NO caused the absorption at 600 nm to decrease in the case of oxidized ceruloplasmin and to increase when starting from the reduced proteins. In this latter case the absorbance at 330 nm was also restored, indicating that NO was able to reoxidize the reduced protein. In all cases the band at 600 nm leveled to ca. 60% of the intensity of the native untreated protein, and new bands below 500 nm appeared in the spectra. While the blue absorption band was restored by removal of NO, the absorbance below 500 nm remained higher even after dialysis. The EPR spectrum resulting from reaction of NO with either oxidized, partially reduced, or fully reduced ceruloplasmin consisted in all cases of a broad, structureless resonance around g = 2. NO caused the reversible disappearance of the type 1 copper EPR spectrum in oxidized ceruloplasmin. Also, the transient novel copper signal that arises during the anaerobic reduction process by ascorbate completely disappeared in the presence of NO and did not reappear upon removal of the gas.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dolphin ceruloplasmin: the first proteolytically stable mammalian ceruloplasmin.

1. Ceruloplasmin, the blue protein of the plasma of vertebrates, was isolated from dolphin, a marine mammal. The protein showed overall physico-chemical parameters very similar to those of all other mammalian ceruloplasmins. The spectroscopic properties indicated a conservation of the copper binding sites. 2. Non-denaturing electrophoresis revealed a conformation similar to that of other mammalian ceruloplasmins. EPR spectroscopy and calorimetric analyses indicated a three-domain arrangement of the protein typical of "aged" ceruloplasmin. 3. Dolphin ceruloplasmin is the only mammalian ceruloplasmin insensitive to trypsin, plasmin or chymotrypsin. This property, however, does not result in a higher conformational stability of the molecule. Thus, susceptibility of ceruloplasmin to aging is not directly related to the lability to proteases, which is typical of all other mammalian ceruloplasmins so far studied.     

Presence of reduced type 1 copper in ceruloplasmin as revealed by reaction with hydrogen peroxide

The reaction of hydrogen peroxide with ox or sheep ceruloplasmin leads to approximately 10% increase of the optical absorption band at 610 nm and of the Type 1 EPR signal. No inactivation or denaturation of the protein is apparent up to 15 H2O2 molar excess. Oxygen is able to restore about 50% of the Type 1 copper absorption in ascorbate-reduced ceruloplasmin, while the other half is recovered after addition of H2O2. It appears that H2O2 undergoes a specific redox reaction with ceruloplasmin, which reveals a fraction of the total copper to be present in the native protein as reduced copper. This fraction is apparently Type 1 copper, while Type 2 is not affected by H2O2.     

Presence of coupled trinuclear copper cluster in mammalian ceruloplasmin is essential for efficient electron transfer to oxygen

The reactivity with dioxygen of a mammalian (sheep) ceruloplasmin, anaerobically reduced with ascorbate, was found to depend on the state of the Type 2 and Type 3 copper centers, as monitored by EPR and optical spectroscopy. A complete reoxidation by air after anaerobic reduction with ascorbate was observed with samples (A) purified by the single-step procedure described for chicken ceruloplasmin (Calabrese, L., Carbonaro, M., and Musci, G. (1988) J. Biol. Chem. 263, 6480-6483), while samples prepared by traditional multistep procedure (B) or subjected to freeze-thawing (C) displayed partial and very slow reoxidation, reflecting the functional nonequivalence of blue coppers which is considered a typical property of mammalian ceruloplasmin. The rate of reduction of the 330 nm chromophore was found to increase as a function of the extent and rate of reoxidation of different samples, while the 610 nm band displayed an opposite trend. Samples B and C showed a Type 2 copper signal in the EPR spectrum, while sample A showed practically no Type 2 copper in the oxidized protein, and a transient Type 2-like signal during reduction. The presence of a trinuclear Type 2-Type 3 cluster can therefore be proposed for all ceruloplasmins, and the integrity of the copper-copper coupling is essential for efficient oxidase behavior.     

Distinction of the two type I coppers in bovine ceruloplasmin

Redox potentials of the two type I copper ions, ‘blue copper ions’, of bovine ceruloplasmin (ferroxidase, iron(II): oxygen oxidoreductase, EC 1.16.3.1) were determined to be 370 and 390 mV (vs. NHE). These two type I copper ions were clearly differentiated during the anaerobic reduction process of oxidized ceruloplasmin and the reoxidation process of completely reduced ceruloplasmin by using absorption, circular dichroic and electron paramagnetic resonance spectroscopies. One of the blue copper ions is reduced faster and reoxidized very slowly, and is assumed to be located away from the active site of ceruloplasmin. On the other hand, the other blue copper ion, which is reduced more slowly and reoxidized rapidly, is supposed to interact with other types of coppers, such as type II (non-blue) and type III (EPR undetectable) coppers. The active site of ceruloplasmin is considered to be comprised of one type I, one type II and a pair of type III copper ions.     

Purification and partial characterization of camel (Camelus Dromedarius) ceruloplasmin

Adult and young camel ceruloplasmin (Cp) were isolated and purified using the single-step chromatography on amino ethyl-activated sepharose. There are no differences between the adult and the young camel protein. The molecular mass of the protein, as estimated by SDS-PAGE (denaturant conditions), was approximately 130000 Da. The electrophoretic mobility of camel Cp is slightly higher as compared to human and sheep protein suggesting that the camel Cp is homogeneous, compact and more acid. The copper content was estimated to be 5.8+/-0.3 atoms per molecule. The spectroscopic feature includes an absorption maximum at 610 nm, which could be attributed to type 1 copper. The EPR spectrum was completely devoid of any typical signal of the type 2 copper. The kinetic parameters of the adult camel Cp for the specific activity as p-phenylendiamine oxidase were determined as K(m)=0.42 mM and V(max)=0.93 microM NADH/mn/mg Cp. The optimum pH for the activity was 5.7.     

Modulation of the redox state of the copper sites of human ceruloplasmin by chloride

Incubation of human ceruloplasmin with physiological concentrations of chloride at neutral pH invariably caused dramatic changes of both the spectroscopic and the functional properties of the protein. The optical intensity at 610 nm increased up to 60%, with a concomitant decrease at 330 nm and the appearance of new bands between 410 and 500 nm. Signals previously undetectable appeared in the EPR spectrum. On the basis of computer simulations, they were interpreted as stemming from an oxidized type 1 copper site and from a half-reduced type 3 copper pair. Removal of chloride completely restored the original optical and EPR lineshapes. Hydrogen peroxide, added to ceruloplasmin in the presence of chloride, was able to capture the electron of the half-reduced type 3 site and to yield a protein insensitive to subsequent removal and readdition of the anion. As a whole, the spectroscopic data indicate that a blue site is partially reduced in the resting protein and that, upon binding of chloride, human ceruloplasmin undergoes a structural change leading to displacement of an electron from the reduced type 1 site to the type 3 site pair. Chloride dramatically affected the catalytic efficiency of human ceruloplasmin. At neutral pH, the anion was an activator of the oxidase activity, being able to enhance up to tenfold the catalytic rate. AtpH < 6, in line with all previous reports, chloride strongly inhibited the activity. At intermediate pH values, i.e., around 6, the effect was composite, with an activating effect at low concentration and an inhibitory effect at higher concentration. Since chloride is present at very high concentrations in the plasma, these results suggest that human ceruloplasmin is, in the plasma, under control of this anion.     

Purification and partial characterization of goose ceruloplasmin

The preparation and properties of ceruloplasmin from goose blood plasma are described. Ammonium sulfate was used to precipitate the crude protein followed by adsorption and elution from DEAE-Sephadex A-50. Further treatment with an ethanol-chloroform mixture and Sephadex G-200 yielded an intensely blue protein possessing a high degree of chemical purity and biological activity. Goose ceruloplasmin, existing in two forms, appears to be a single polypeptide, apparent Mr121,300, with an A610/A280 ratio of 0.07. Copper represented 0.32%, which corresponded to six atoms of copper per protein molecule. Although the amount of EPR-detectable copper was the same as in mammalian ceruloplasmins there were some differences in EPR parameters, mainly in A parallel. Goose ceruloplasmin's amino acid composition, although similar in many residues to human ceruloplasmin, was lower in tyrosine, cystine/cysteine, and acidic amino acids. Valine was found as the N-terminal amino acid. Hexose, hexosamine, sialic acid, and fucose accounted for 6.65% of the weight. Goose protein contained only half the sialic acid of human ceruloplasmin. Two values for Km using either p-phenylenediamine (0.64 and 0.053 mM) or o-dianisidine (0.76 and 0.15 mM) were evaluated from Lineweaver-Burk plots. EPR studies on reactions with water radiolysis products at cryogenic temperatures allowed us to discover that goose ceruloplasmin, like human and bovine ceruloplasmins, possesses superoxide dismutase activity.     

The interaction of nitric oxide with ascorbate oxidase.

1. The reaction of nitric oxide with oxidized and reduced ascorbate oxidase (L-ascorbate: oxygen oxidoreductase, EC 1.10.3.3) has been investigated by optical absorption measurements and electron paramagnetic resonance, and the results are compared with those of ceruloplasmin. 2. Upon anaerobic incubation of oxidized ascorbate oxidase with nitric oxide a decrease of the absorbance at 610 nm is found, which is due to an electron transfer from nitric oxide to Type-1 copper. 3. In the presence of nitric oxide the EPR absorbance of ascorbate oxidase decreases and shows predominatly a signal with characteristics of Type-2 copper (g parallel = 2.248; A parallel = 188 G), whereas the type-1 copper signal has vanished. 4. Comparison of the intensities of the EPR signals before and after NO-treatment points to the presence of one Type-2 and three Type-1 copper atoms per molecule of ascorbate oxidase. 5. It is shown that the changes in the optical and the EPR spectrum of ascorbate oxidase induced by nitric oxide are reversible. No difference in enzymic activity is found between the native enzyme and the NO-treated enzyme after removal of nitric oxide.     

The type 2 copper of ascorbate oxidase

Ascorbate oxidase, dissolved in Hepes or sodium phosphate buffers, was analyzed by EPR and activity measurements before and after storage at −30°C and 77 K. The specific activity was somewhat higher in the phosphate buffer, about 3500–3700 Dawson units compared to about 3100 units of the enzyme dissolved in Hepes buffer. After storage at −30°C the activity fell to 1400–2000 units in the phosphate buffer but only to 2600–2800 units in the Hepes buffer. Large changes occurred in the EPR spectrum of enzyme dissolved in the phosphate buffer after storing at −30°C suggesting an alteration of the type 2 copper site. These changes were, however, reverted when the samples were thawed and rapidly frozen at 77 K. Copper analysis showed that about 50% of the total copper was EPR detected. The type 2 Cu²⁺ EPR intensity was in most samples close to 25% of the total EPR intensity. This low contribution of type 2 Cu²⁺ could not be changed if the enzyme was completely reduced and reoxidized, treated with Fe(CN)₆³⁻, large excess of NaF, addition of 50% (v/v) ethylene glycol or dialyzed against 0.1 M Mes buffer (pH 5.5). Since the crystal structure shows that there are one each of types 1 and 2 copper in the monomers there must be another species with an EPR signal rather different from these two copper species. This signal is proposed to originate from some trinuclear centers. The EPR simulations show that it is possible to house a broad unresolved signal under the resolved type 1 and 2 signals so that the total integral becomes 50% of the total copper in the molecule.     

A tyrosine-derived free radical in apogalactose oxidase

Oxidation of apogalactose oxidase with ferricyanide leads to the formation of a stable free radical exhibiting distinctive optical absorption and EPR spectral features. The radical is associated with absorption in both near-UV and near-IR spectral regions, and its EPR spectrum is characteristic of an aromatic free radical with gav = 2.005. Reconstitution of both the apoenzyme and the free radical-containing form with copper substantially restores both the absorption spectra and the catalytic activity of the active enzyme, indicating that the preparation of the radical species does not significantly damage the protein. The absence of a free radical EPR signal in reconstituted and activated galactose oxidase containing nearly stoichiometric copper suggests the radical is an active site species relating to the free radical-coupled copper site previously proposed for this enzyme. Isotopic labeling experiments demonstrate that the radical derives from a tyrosine residue. The distinctive spectra associated with this radical indicate an environment which is different from that associated with the tyrosyl phenoxyl sites in other free radical enzymes.     

A cytoplasmic nickel-iron hydrogenase with high specific activity from Desulfovibrio multispirans sp. N., a new species of sulfate reducing bacterium

A hydrogenase from a new species of sulfate reducing bacterium has been isolated and characterized. In contrast to other hydrogenases isolated from Desulfovibrio, this enzyme is found in the cytoplasmic fraction rather than in the periplasm. The specific activity of the enzyme, as measured in the hydrogen evolution assay, is twice as high as the specific activity of the hydrogenase from D. gigas. It also differentiates itself from the periplasmic Desulfovibrio hydrogenases by being more active in the hydrogen evolution rather than in the hydrogen uptake assay. The enzyme was shown to contain 0.9 atoms of nickel, 11 atoms of iron and 10 atoms of labile sulfide per mole of enzyme. It exhibits an unusually low intensity of the g = 2.31 nickel EPR signal in the isolated enzyme but shows a normal intensity for the g = 2.19 nickel EPR signal when reduced under hydrogen.     

Mechanisms of copper incorporation during the biosynthesis of human ceruloplasmin

To examine the mechanisms of copper incorporation during ceruloplasmin biosynthesis, we developed methods to resolve and identify apo and holoceruloplasmin. The identity of holoceruloplasmin was confirmed by oxidase activity staining, immunoblotting, 67Cu-ligand exchange, and 67Cu-ligand blotting. Following metabolic labeling of human liver and lung cell lines with 67Cu, newly synthesized holoceruloplasmin was detected in the culture media as two species with apparent molecular masses of 84 and 79 kDa. Pulse-chase studies demonstrate that exogenous copper is readily available for incorporation into newly synthesized ceruloplasmin and that the kinetics of apo and holoceruloplasmin synthesis and secretion are identical. Inhibition of N-linked glycosylation did not affect the rate or amount of copper incorporated into newly synthesized ceruloplasmin but did result in the secretion of a single 68-kDa holoceruloplasmin moiety. Despite differences in the kinetics of copper uptake between cell lines a linear rate of copper incorporation into newly synthesized ceruloplasmin was observed with no evidence of copper exchange following biosynthesis. Under the conditions studied, holoceruloplasmin accounted for less than 5% of the total ceruloplasmin synthesized and secreted by each cell line. The data indicate that copper is incorporated into newly synthesized ceruloplasmin early in the course of biosynthesis by a process independent of N-linked carbohydrate addition. This process of copper incorporation results in an apparent conformational change in the ceruloplasmin molecule which does not affect the secretory rate of the protein.     

Characterization of a small metal binding protein from Nitrosomonas europaea

A small metal-binding protein (SmbP) with no known similarity to other proteins in current databases was isolated and characterized from the periplasm of Nitrosomonas europaea. The primary structure of this small (9.9 kDa) monomeric protein is characterized by a series of 10 repeats of a seven amino acid motif and an unusually high number of histidine residues. The protein was isolated from N. europaea with Cu(II) bound but was found to be capable of binding multiple equivalents of a variety of divalent and trivalent metals. The protein was overexpressed in Escherichia coli and used for the study of its metal-binding properties by UV/vis, circular dichroism (CD), and electron paramagnetic resonance (EPR) spectroscopy and equilibrium dialysis and isothermal titration calorimetry. The protein was found to bind up to six Cu(II) atoms with dissociation constants of approximately 0.1 microM for the first two metal ions and approximately 10 microM for the next four. Binding of Cu(II) resulted in spectroscopic features illustrating two distinctive geometries, as determined by EPR spectroscopy. The levels of SmbP in the periplasm were found to increase by increasing the levels of copper in the growth media. This protein is proposed to have a role in cellular copper management in the ammonia-oxidizing bacterium N. europaea.     

Ceruloplasmin in human plasma and its relationships with the copper-albumin complex.

The electron paramagnetic spectrum of human plasma is dominated, in the g = 2 region, by resonances from copper atoms bound to ceruloplasmin, and does not reveal the fraction of copper normally associated with albumin, except in a few cases, where a copper-albumin signal increases with time after blood withdrawal. This copper-albumin complex is responsible for a resonance at a g value below g = 2 in the spectrum of human serum, which has been recently attributed to a modified form of type 2 copper bound to ceruloplasmin [Rylkov, V.V., Tarasiev, M.Y. & Moshkov, K.A. (1991) Eur. J. Biochem. 197, 185-189]. In the plasma, copper associated to albumin comes from ceruloplasmin. Purified ceruloplasmin is unable to exchange copper with albumin, either purified or in plasma. It can not be ruled out that some serum components trigger the metal exchange, in a defence mechanism operating when ceruloplasmin leaks, by unknown processes, its copper content before discharging the metal into the various organs.     

Reaction of human ceruloplasmin and anion treated ceruloplasmin with diethyldithiocarbamate

The reaction of human ceruloplasmin and anion treated ceruloplasmin with diethyldithiocarbamate was studied at pH 5.5. The analysis of optical and EPR spectra at 9 GHz showed that ceruloplasmin contains five paramagnetic copper ions, two of which, X and Y, not involved in enzymatic activity, are chelated by diethyldithiocarbamate; the complex thus formed is easily removed by high-speed centrifugation. However, the enzyme depleted of these two X and Y copper ions is able to compete with the Cu(II)-diethyldithiocarbamate complex, as time elapses, recovering both Cu(II) atoms. In addition diethyldithiocarbamate acts as a reducing agent for the two type-I copper atoms when added in large excess to the enzyme or the anion treated enzyme.     

Electron paramagnetic resonance study of storage effects on ceruloplasmin in human serum compared with purified ceruloplasmin in aqueous solution

The EPR signal amplitude of human serum ceruloplasmin shows significant changes as a function of time and temperature during storage. The same behavior occurs with aqueous solutions of purified ceruloplasmin. From the observation that the spectral lines of the EPR signal of ceruloplasmin from unmanipulated serum are identical to those coming from purified ceruloplasmin, we conclude that only type I Cu2+ of ceruloplasmin are involved in the signal changes. A temperature-dependent electron shift toward type I Cu2+ paramagnetic centers, occurring via the type II and type III Cu2+ species of the protein, is believed responsible for the process. The possible origin of the reducing electrons is discussed. A procedure to obtain reproducibility of recording of EPR spectra of ceruloplasmin in physiological fluids is proposed.     

Electron paramagnetic resonance study of water distribution in starch granules

An electron paramagnetic resonance (EPR) study was performed for potato and wheat starch containing Cu2+ ions as a paramagnetic probe. Distribution of water in the starch granules as well as the interactions between the copper and starch matrix of different crystalline structures were determined. EPR spectra of the native starches consisted of two different centers of Cu2+. One of them, giving at 293 and 77 K an EPR signal of axial symmetry with a well-resolved hyperfine structure (HFS), was assigned to the Cu2+ -starch complex in which Cu2+ ions strongly interacted with oxygen atoms of the starch matrix. Another Cu2+ species, exhibiting an isotropic signal at 293 K and an axial signal with resolved HFS at 77 K, was attributed to a [Cu(H2O)6]2+ complex freely rotating at room temperature and immobilized at low temperatures. Interaction of Cu2+ with the starch matrix and the relative number of the particular copper species depended on the crystallographic type of starch. Dehydration at 393 K resulted in elimination of the rotating complex signal and decrease of the total intensity of the EPR spectrum caused by clustering of the Cu2+ ions. Freezing at 77 K and thawing led to restoring of the spectrum intensity and reappearing of the signal of the [Cu(H2O)6]2+ complex. This effect, related to liberation of water molecules from the granule semicrystalline growth rings on freezing/thawing, was especially visible for wheat starch, indicating differences in the water retention ability of starch granules of different crystallographic structure.