A novel form of copper-containing centers in human ceruloplasmin

Using spectral methods (EPR, spectrophotometry), it was demonstrated that type II Cu2(+)-centers (so-called non-blue centers) are represented in human ceruloplasmin by two (but not one) stable forms which differ in their EPR spectra and absorption properties. Differential spectra were recorded, and the difference in the extinction coefficients of these forms was determined. Both forms were detected by the EPR method in blood sera from healthy and diseased individuals. The relative amount of these forms depends on the origin of the disease. This finding opens new perspectives in the diagnostic application of the EPR method. Spectrophotometric evidence of the ferroxidase activity of serum ceruloplasmin towards hemoglobin was obtained; other serum components were also shown to be involved in this process.     

Labile conformation of type 2 Cu2+ centres in human ceruloplasmin.

1. The investigation of human ceruloplasmin by spectral methods (EPR and spectrophotometry) demonstrated that type 2 Cu2(+)-containing centres occur not in one, but in two stable forms, differing in EPR and optical spectra. The differential optical spectra of these forms were recorded and the differences in molar absorption coefficients determined. 2. By the EPR method, it was shown that both forms of these centres exist in the blood serum of control donors, as well as in the serum of patients. The relative content of these forms depends on the organism physiological state or on the presence of some pathological condition. 3. The ferroxidase activity of ceruloplasmin against hemoglobin was proved spectrophotometrically. The involvement of other serum proteins in this process cannot be ruled out. The conformational state of ceruloplasmin molecules plays an essential role in its oxidase activity.     

The influence of radiotherapy on ceruloplasmin and transferrin in whole blood of breast cancer patients

Ceruloplasmin and transferrin are proteins which play a potential role in the process of breast cancer development. These molecules contain Cu²⁺ (ceruloplasmin) or Fe³⁺ ions (transferrin) and thus constitute paramagnetic centers, which can be studied using electron paramagnetic resonance method. The aim of the study was to determine how paramagnetic centers in whole blood of breast cancer patients change under the influence of radiation therapy. Samples of whole blood were taken from 17 women with breast cancer treated with radiotherapy. The measurements were carried out at 170 K using X-band electron paramagnetic resonance (EPR) spectrometer Bruker EMX-10. Two distinct EPR lines, derived from high-spin Fe³⁺ in transferrin and Cu²⁺ from ceruloplasmin, were revealed in all frozen samples. The amplitude and integrated intensity of the EPR signal from Cu²⁺ in ceruloplasmin significantly decreased in all patients after the delivery of the radiation fraction. When comparing the integral intensity of the signal from Fe³⁺ in transferrin, three different situations were identified which are patient specific: a significant increase, an insignificant change, or a significant decrease after the irradiation. A decreased level of Cu²⁺ from ceruloplasmin in patients after radiotherapy means a low level of ceruloplasmin in the plasma or an increased content of reduced Cu⁺ ions. Differences in the integrated intensity of the EPR signal from transferrin translate directly into the amount of bound iron. The observed changes could indicate how well the organism fights against cancer and how easily it adapts to the situation of biochemical stress.     

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.     

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.     

The effect of some anions on the spectral properties of bovine ceruloplasmin

The effect of binding of N3-, SCN-, OCN-, and F- to bovine ceruloplasmin (Cp) has been studied in detail using absorption, circular dichroic (CD), and electron paramagnetic resonance (EPR) spectroscopies. With the addition of increasing amounts of N3-, SCN-, and OCN- to a Cp solution, the intensity of the band at 614 nm at first increased several percent and then decreased gradually as at least one type I copper was reduced and/or as the type I copper was changed to type II copper. Concomitantly, new bands appeared at 430 and 365 nm for N3-, 435 and 380 nm for SCN-, and about 390 nm for OCN-. A conformational change in the protein induced by the binding of N3-, SCN-, and OCN- to the type II and type III coppers led to the change in the CD spectra. The observed increase of the band at about 430 nm was attributed to the change occurring at the type I copper site. On the other hand, the band at about 370 nm may come from a charge transfer of coordinated anions to the Cu(II) ion. Fluoride ion did not induce the appearance of the band at around 430 and 370 nm, but the parallel component of the type II copper EPR signal was split upon the binding of two fluoride ions to the copper ion.     

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.     

Allosteric regulation of ceruloplasmin activity]

Study of the interactions of homogenous human ceruloplasmin preparations with histamine show that the rate of p-phenylene diamine oxidation by ceruloplasmin is increased in the presence of histamine; the increase in the enzyme activity is independent of histamine concentration. The dependence of the reaction rate on substrate concentration is S-shaped, both in the presence and in the absence of histamine. The respective values of the Hill coefficient and Rs for the enzyme in the presence and in the absence of histamine are 2.5 and 2.0 and 8.0 and 10.4. Histamine does not change ceruloplasmin-specific absorption at 610 nm. Evidence from EPR studies show that histamine does not interact with Cu of the enzyme active center. During interaction with histamine the antigenic properties of the enzyme are changed. Histamine increases the oxidase activity of the enzyme in human and rat blood sera and exerts multifold effects on the enzyme activity in patients with hepatolenticular degeneration. After injection of histamine to rats the enzyme activity is increased without a simultaneous increase in Cu concentration in the blood serum, i.e. without de novo synthesis of ceruloplasmin. The data obtained suggest that ceruloplasmin is probably an allosteric enzyme, which histamine is its positive allosteric effector.     

Type 2 Cu2+ in pMMO from Methylomicrobium album BG8

EPR spectra were obtained for the type 2 Cu2+ site in particulate methane monooxygenase (pMMO) from Methylomicrobium album BG8 grown on K15NO3 and 63Cu(NO3)2. The concentration of the type 2 Cu2+ signal was approximately 200 microM per 25 mg/ml protein in packed cells and membrane fractions, a concentration that is consistent with its attribution to pMMO, and the EPR parameters were consistent with electron paramagnetic resonance (EPR) parameters previously assigned to pMMO. The superhyperfine structure due to nitrogen is better resolved because I = 1/2 for 15N whereas I = 1 for 14N and A(15N)/A(14N) = 1.4. Under these conditions, superhyperfine structure is resolved in the g region of the X-band spectrum. At low microwave frequency (S-band) the resolution of the nitrogen superhyperfine structure improves. Signals are attributed to type 2 Cu2+ in which cupric ion is bound to four (less likely three) nitrogen donor atoms.     

A new copper(II) electron paramagnetic resonance signal in two laccases and in cytochrome c oxidase

A new EPR signal from Cu2+ has been discovered in reductive experiments with type 2 copper-depleted laccase from Polyporus versicolor. A novel EPR signal has also been found in native laccase from Rhus vernicifera on oxidation of the reduced protein with H2O2. In reoxidation experiments with cytochrome c oxidase from beef heart, a new Cu2+ signal has been observed. With Rhus laccase, the new signal is shown to originate from one of the copper ions that are nondetectable in the resting enzyme, and evidence is presented for the signals in Polyporus laccase and cytochrome c oxidase also stemming from the metal pairs that are antiferromagnetically coupled in the oxidized enzymes. The new signals show strong rhombic character, and the EPR parameters place them in a category different from the signals of type 1 as well as of type 2 Cu2+ ions.     

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.     

Spectral characteristics of the mechanism of oxidase activity of ceruloplasmin

The absorbance and EPR spectra of type 1 and 2 copper-binding centres which are present in ceruloplasmin (Cp) molecule were shown to disappear upon the reduction of the enzyme by ascorbate under anaerobic conditions. The fluorescence band attributed to type 3 Cu was altered concomitantly. The electron-accepting nitroxyl radical added to reduced Cp restored the absorbance, EPR and fluorescence spectra of the oxidase. Only type 1 and 3 copper ions, as judged by spectral changes, can be reduced by ascorbate and then reoxidized by the nitroxyl radical in the azide-treated Cp. The spectral properties of Cp provided by copper ions of different types change simultaneously and concordantly upon oxidation/reduction. This seems to be caused by cooperative interaction of these ions involved in the electron transfer from the donating substrate to the accepting molecule of the nitroxyl radical (in model studies of oxidase reaction) or oxygen (under natural conditions). The copper ions in the active centre of Cp constitute an intramolecular electron transport chain, which may, at least in vitro, function without one of its links.     

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)     

On the lability and functional significance of the type 1 copper pool in ceruloplasmin

 The possibility that ceruloplasmin (CP) functions as a copper transferase has fueled a continuing interest in studies of the copper release process. The principal goal of the current investigation has been to identify the most labile copper centers in sheep protein. In fact, subjecting the enzyme to a slow flux of cyanide at pH 5.2 under nitrogen in the presence of ascorbate and a phenanthroline ligand produces partially demetalated forms of the protein. By standard chromatographic techniques it is possible to isolate protein with a Cu/CP ratio of ∼4 or ∼5 as opposed to the native protein which has Cu/CP=5.8. In contrast to other blue oxidases, analysis suggests that CP preferentially loses its type 1 coppers under these conditions. Thus, the spectroscopic signals from the type 1 centers exhibit a loss of intensity while the EPR signal of the type 2 copper becomes stronger. Furthermore, the Cu/CP≈4 and Cu/CP≈5 components retain about 50% of the activity of the native protein, consistent with an intact type 2/type 3 cluster. All three type 1 copper sites appear to suffer copper loss. Reconstitution with a copper(I) reagent restores the spectroscopic properties of the native protein and 90% of the original activity. The results suggest a possible functional significance for the presence of three type 1 coppers in CP. By employing a pool of redox-active but relatively labile type 1 copper centers, the enzyme can serve as a copper donor, if necessary, without completely sacrificing its oxidase activity. Received: 15 February 1999 / Accepted: 22 April 1999     

Magnetic susceptibility of laccases and ceruloplasmin.

1. Recent magnetic susceptibility measurements on laccase (monophenol,dihydroxyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1) from the lacquer tree Rhus vernicifera showed a deviation from Curie behaviour above 50 K, which was taken as evidence for an antiferromagnetically coupled Cu(II)-Cu(II) pair in the oxidized enzyme. The magnetic susceptibility of this protein has been reinvestigated. Further measurements on laccase from the fungus Polyporus versicolor and human ceruloplasmin (iron(II):oxygen oxidoreductase, EC 1.16.3.1) are presented. 2. The magnetic susceptibility of fungal laccase and lacquer tree laccase can be accounted for by the EPR detectable copper ions in the temperature range 40--300 K. 3. If an antiferromagnetically coupled Cu(II)-Cu(II) pair exists in the laccases, then the coupling, expressed as --J, should be at least of the order of 300 cm-1, as deduced from the Curie dependence of the susceptibility and the sensitivity in our measurements. 4. If an analogy with the laccases is assumed for the EPR invisible copper in ceruloplasmin then a limiting value of the coupling may be deduced also in this case, with --J at least of the order of 200 cm-1.     

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.     

Paramagnetic probes of the domain structure of histidine-rich glycoprotein

The interaction of Cu2+ and Fe3+-mesoporphyrin with histidine-rich glycoprotein (HRG) from rabbit serum was examined spectroscopically. The first equivalent of Cu2+ binds to HRG producing a type II electron paramagnetic resonance (EPR) spectrum with g[[ = 2.25, gm = 2.05, A[[ = 0.019 cm-1 (180 G), and superhyperfine along gm. These spectral parameters suggest moderately covalent coordination of Cu2+ to the protein by nitrogens. With increasing Cu2+ the superhyperfine disappears; however, the g and A values change only marginally. The increase in EPR signal amplitude throughout the addition of 1-15 equiv of Cu2+ is linear and thereafter maximizes, suggesting 18-22 equiv are bound. In contrast, changes in the circular dichroism spectrum at 280 nm appear sigmoidal and can be interpreted as the binding of Cu2+ to two structurally distinct regions of the protein. Evidence for two structurally distinct binding domains is found by comparing EPR spectra of Cu2+ complexes of HRG with spectra from complexes of two of its major proteolysis products (peptides). After binding 1 equiv of Cu2+, both the 30-kDa histidine-rich peptide and the native protein exhibit identical spectra including the pronounced superhyperfine. In contrast, the spectrum of the histidine-normal 45-kDa peptide with 1 equiv of Cu2+ bound lacks superhyperfine and parallels closely that of the native protein with 20 equiv bound. Finally, Fe3+-mesoporphyrin binds to HRG exhibiting both high-spin (g = 6.05) and low-spin (gz = 2.94, gy = 2.25, gx = 1.50) EPR resonances, and the latter imply bis(histidine) coordination.(ABSTRACT TRUNCATED AT 250 WORDS)     

The identity of a new copper(II) electron paramagnetic resonance signal in cytochrome c oxidase

In reoxidation experiments with cytochrome c oxidase (EC 1.9.3.1) in the presence of both reducing substrate and molecular oxygen, a new EPR signal from Cu2+ has been observed. The new signal corresponds to 0.45 Cu per functional unit. It is concluded that the new EPR signal originates from CuB2+, the copper which is EPR-nondetectable in the resting enzyme. Optical absorption changes in the 500-700 nm region accompanies the decay of the new Cu2+ EPR signal. Based on the results in this investigation a catalytic cycle for cytochrome oxidase is proposed.     

EPR study of serum ceruloplasmin and iron transferrin in myocardial infarction.

Electron paramagnetic resonance (EPR) analysis of frozen serum from myocardial infarction patients has been conducted. Signal at g=4.3 was found definitively attributable to iron(III)-transferrin complex. Imcrease of serum ceruloplasmin as compared to normal was confirmed, with a concomitant decrease of iron-transferrin content. A mechanism for such correlated variation is hypothesized.