Aceruloplasminemia is a rare autosomal recessive iron overload disease caused by mutations in the gene of the ferroxidase ceruloplasmin. Based on the X-ray structure of human ceruloplasmin, attempts were made to interpret the aceruloplasminemia

The review presents both our own and literature data on studies of pathways of evolution of the so-called multinuclear blue copper-proteins (MBCP) that have the domain organization. The MBCP are widely spread in living nature, they have been revealed in cells of archei, bacteria, and eukaryotes. The MBCP composition includes the copper-proteins such different by their properties as oxidases, reductase, blood coagulation factors V and VIII. Most likely, MBCP have been originated from a low-molecular protein-precursor similar topologically with the blue electron-transporting protein of the cupredoxin type, as a result of action of various evolutionary mechanisms: amplification of genes, formation of protein structures by different combinations of domains, a change of size of domains, the segment elongation at the expense of the activational domain, formation and loss of copper-binding centers, variation of amino acid ligands in such centers, the appearance of centers of binding of other proteins, glycosylation, etc.

Authors review the results of their research and data available from literature on the mechanism of ferroxidase activity of copper-containing human plasma enzyme ceruloplasmin (Fe(II): oxygen-oxidoreductase; EC 1.16.3.1). The described mechanism is based on the analysis of structural data and suggests that ceruloplasmin catalyzes consecutive transfer of 4 electrons from substrate to О2 molecule which, by virtue of four-electron reduction of О-О bond, is then converted into Н2О molecule according to the scheme: 4Fe2++O2+4H+=2H2O+4Fe3+. The process of intramolecular electron transfer proceeds from oxidized Fe2+ions along the chain of copper binding sites from mononuclear center T1Cu to trinuclear cluster, consisting of T2Cu center and dinuclear center T3Cu (site of О2 reduction to Н2О). Ions Fe2+ are localized in labile binding centers (M2+centers of binding bivalent metal ions), located in proximity of T1Cu centers. Formed in the oxidation reaction, Fe3+ ions are further transported to M3+ centers of binding trivalent metal ions located in close proximity to the protein surface. As a result of subsequent transport from ceruloplasmin globule, Fe3+ becomes capable of forming complexes with apo-transferrin.

The X-ray structure of human serum ceruloplasmin has been solved at a resolution of 3.1 Å. The structure reveals that the molecule is comprised of six plastocyanin-type domains arranged in a triangular array. There are six copper atoms; three form a trinuclear cluster sited at the interface of domains 1 and 6, and there are three mononuclear sites in domains 2, 4 and 6. Each of the mononuclear coppers is coordinated to a cysteine and two histidine residues, and those in domains 4 and 6 also coordinate to a methionine residue; in domain 2, the methionine is replaced by a leucine residue which may form van der Waals type contacts with the copper. The trinuclear centre and the mononuclear copper in domain 6 form a cluster essentially the same as that found in ascorbate oxidase, strongly suggesting an oxidase role for ceruloplasmin in the plasma.

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.

Conformational properties of ceruloplasmin were studied immediately in blood serum of healthy volunteers, patients with tuberculosis, with pulmonary cancer, with pneumonia and with Wilson-Konovalov disease. The glycoprotein conformation was found to depend on the volunteer physiological state and/or available pathology. The ceruloplasmin conformation and status of its copper-containing sites of the I type affected the enzyme oxidase properties and hence routine colorimetric procedures require some corrections for estimation of ceruloplasmin concentration and activity in blood serum.

Conformational properties of ceruloplasmin were studied immediately in blood serum of healthy volunteers, patients with tuberculosis, with pulmonary cancer, with pneumonia and with Wilson-Konovalov disease. The glycoprotein conformation was found to depend on the volunteer physiological state and/or available pathology. The ceruloplasmin conformation and status of its copper-containing sites of the I type affected the enzyme oxidase properties and hence routine colorimetric procedures require some corrections for estimation of ceruloplasmin concentration and activity in blood serum.

The changes in metabolism of biogenic amines and beta-endorphine by a new effective method of alcoholism narcopsychotherapy induced by ketamine administration were investigated. It was shown that during an experiment monoaminoxidase (MAO) activity (type B) in blood platelets and MAO (type A) activity in blood serum are decreased, ceruloplasmin activity is raised, dopamine and beta-endorphine concentrations in blood are increased, whereas serotonin content is not changed. It is shown that the inhibition of MAO-B activity by ketamine has a noncompetive character. The possible role of the changes in metabolism of biogenic amines in hallucinogenic ketamine effects and also their importance for efficiency of dependence treatment on alcohol are discussed.

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.

A new narcopsychotherapeutic technique termed "affective counterattribution" (ACA) is offered to treat alcohol addiction. The use of ACA increased the effectiveness of alcoholism treatment that was ensured by association of strong pharmacogenic negative emotional experience coupled with bright hallucinatory images to notions of alcohol and alcohol-related stimuli. Alcoholic attitude was destroyed and the patients' pathological personality traits were corrected.

The identification of possible copper ligands in human ceruloplasmin was carried out by the computer similarity analysis for sequences of ceruloplasmin and several other copper oxidases: azurin, plastocyanin, superoxide dismutase, tyrosinase and hemocyanin. It follows from the analysis of inter- and intramolecular homology that copper active sites of different types appeared to be in close contacts within the ceruloplasmin molecule.