10-(6'-Plastoquinonyl)decyltriphenylphosphonium (SkQ1) Does Not Increase the Level of Cytochromes P450 in Rat Liver and Human Hepatocyte Cell Culture.

Mitochondria-targeted antioxidant SkQ1 did not increase the content of cytochromes P450 in livers of rats that were given SkQ1 in drinking water for 5 days in a dose (2.5 µmol per kg body weight) that exceeded 10 times the SkQ1 therapeutic dose. SkQ1 did not affect the levels of cytochrome P450 forms CYP1A2, CYP2B6, and CYP3A4 in monolayer cultures of freshly isolated human hepatocytes, while specific inducers of these forms (omeprazole, phenobarbital, and rifampicin, respectively) significantly increased expression of the cytochromes P450 under the same conditions. We conclude that therapeutic doses of SkQ1 do not induce cytochromes P450 in liver, and the absence of the inducing effect cannot be explained by poor availability of hepatocytes to SkQ1 in vivo.

Effect of Long-Term Treatment with Antioxidant SkQ1 Added to Drinking Water on Cytochromes P450 Level in Rat Liver.

Mitochondria-targeted cationic antioxidant plastoquinonyl decyltriphenylphosphonium (SkQ1) added to drinking water in therapeutic doses (250 nmol/kg per day) for a long time (up to 24 months) does not induce cytochromes P450 in rat liver.

Therapeutic doses of SkQ1 do not induce cytochromes P450 in rat liver.

The effect of SkQ1 (a mitochondria-targeted antioxidant) on the level of cytochromes P450 in rat liver was studied. It was found that administration of therapeutic dose of SkQ1 with drinking water for 5 days (250 nmol/kg of body weight per day) did not alter the level of cytochromes P450. Under the same conditions, the standard dose of phenobarbital used for the induction of cytochromes P450 caused the 2.7-fold increase in the content of these cytochromes. We conclude that therapeutic doses of SkQ1 do not induce cytochromes P450 in rats.

Conformational changes near the cytochrome P450 active site upon binding of two different ligands.

It is shown that a stable nitroxyl radical, 4-cyano-2,2,6,6-tetramethylpiperidine-1-oxyl, forms a complex with cytochrome P4502B4 by analogy with the second type substrates by joining directly to pentacoordinate heme iron. The bound radical is inaccessible to water-soluble paramagnetic ions, which confirms its localization in a hydrophobic pocket near the heme. Benzphetamine and N,N-dimethylaniline, the first-type nonpolar substrates, induce conformational changes of the spin-labeled hemoprotein which are evidently accompanied by an increase in the volume of the pocket resulting in emergence of contact with aqueous phase, and the heme-bound spin label becomes accessible to water-soluble paramagnetics. In this case potassium ferricyanide broadens the spin-labeled cytochrome signal and, as a result, lowers the amplitudes of the spectral components. Similar changes were registered at non-micellar concentrations of nonionic detergent Emulgen 913, whose activating effect on hydroxylation reactions is associated, as we showed previously, with its presence in the CYP2B4 active site simultaneously with substrates.

New findings in studies of cytochromes P450.

Cytochromes P450 represent a numerous family of heme-containing enzymes belonging to the group of monooxygenases. In prokaryotes, cytochromes P450 usually perform a plastic function, whereas in eukaryotes their functions are very diverse. Mammalian cytochromes P450 are components of membranes and are involved in biosynthesis and metabolism of many physiologically active substances; moreover, these cytochromes are unique in their ability to catalyze biotransformation of xenobiotics, i.e. metabolize substances of foreign origin (drugs, toxins, environmental pollutants). The latter promotes elimination of xenobiotics, but sometimes intermediates of their metabolism are even more toxic and dangerous than the original xenobiotics per se. Some catalytic features of cytochromes P450 still need unambiguous explanation, i.e. broad substrate specificity, diversity of catalytic reactions, and unusual kinetics. Under some conditions, cytochromes P450 can produce reactive oxygen species, and this is another problem attracting increasing attention. In this respect, a recent finding in mitochondria of analogs of microsomal cytochromes P450 seems especially intriguing; it was postulated that P450 can be responsible for mitochondrial dysfunction, cell apoptosis, and pathogenesis of some diseases. In this paper the present state of the art concerning these problems is considered.

Cross-Linking study of cytochrome P450 1A2 in proteoliposomes.

Proteoliposomes, containing cytochrome P450 1A2, were obtained by the cholate-dialysis technique. The effect of bifunctional cross-linking reagents on the purified hexameric cytochrome P450 1A2 in an aqueous medium and on the proteoliposomal P450 1A2 have been compared. Electrophoretic analysis of the modified proteins demonstrated the same oligomeric (hexameric) organization of the hemoprotein in each case.

Cytochrome P450 1A2: oligomers in proteoliposomes.

The presence of oligomers of cytochrome P450 1A2 in membranes of proteoliposomes produced by the cholate-dialysis technique was demonstrated by cross-linking of protein molecules with bifunctional reagents followed by electrophoretic analysis of the modified proteins. A hexameric organization of cytochrome P450 1A2 in the membrane of proteoliposomes is suggested with high probability based on the comparison of the purified hemoprotein oligomeric structure in an aqueous medium and that in the proteoliposomes. The comparison was carried out using the same method.

Specificity of self-assembly of cytochrome P450.

Under certain conditions, hexamers of microsomal cytochrome P450 can self-assemble from the subunits of different isoforms. However, the possibility for free choice results in recognition between identical subunits of each form of cytochrome P450 which provides preferential association of identical monomers into corresponding hexamers. The specificity of self-assembly suggests hexameric arrangement of cytochrome P450 in native membranes as we proposed earlier. In the present study, highly purified cytochrome P450 2B4 and cytochrome P450 1A2 (CYP 2B4 and CYP 1A2), including those immobilized by covalent attachment to an insoluble carrier of one protomer of each hexamer, were employed.

Cytochrome P-450: hexameric structure of the purified LM4 form.

Purified cytochrome P-450LM4 was found to be monodisperse in 20% glycerol by analytical ultracentrifugation. Its S20,w value was quite similar to that of hexameric P-450LM2. At lower glycerol concentrations the P-450LM4 showed a tendency to aggregate. The P-450LM4 oligomers were immobilized on Ultrogel A4 under conditions allowing only one covalent link to the matrix per oligomer. In the presence of SDS, the oligomers dissociated leaving only 1/6th of the initial amount of bound protein on the matrix, suggesting that the purified P-450LM4 is a hexamer. This was confirmed by electron microscopy. The quaternary structure of the P-450LM4 was similar to that demonstrated earlier for P-450LM2.

Cytochrome P-450LM2 oligomers in proteoliposomes.

The effects of bifunctional cross-linking reagents on the purified hexameric cytochrome P-450LM2 in an aqueous medium and on the proteoliposomal cytochrome P-450LM2 have been compared. In both cases, cross-linking is shown to result in the appearance of a range of additional protein bands in SDS electrophoretograms. The number and the positions of these bands seem to be similar in the solubilized and in the proteoliposomal cytochromes. No additional bands appear when the purified cytochrome P-450 was pretreated with 0.2%. Emulgen 913, which decomposes cytochrome P-450LM2 hexamers. The results indicate that the membrane-bound cytochrome P-450 can exist in the oligomeric (presumably hexameric) form.

Immobilized cytochrome P-450LM2. Dissociation and reassociation of oligomers.

Subunit interactions in the purified hexameric cytochrome P-450LM2 have been studied using covalent binding of one of the 6 promoters to an insoluble matrix. High ionic strength, large-scale pH changes, guanidine chloride and sodium cholate taken at membrane-solubilizing concentrations, had no effect on the aggregation state of the immobilized hemoprotein. SDS caused a 6-fold decrease in the amount of the bound cytochrome. Non-ionic detergents (Emulgen 913, octylglucoside, Tritons) induced hexamer dissociation. In the presence of Emulgen 913 (greater than 0.2%), monomers and immobilized dimers were obtained as cytochrome P-450 was studied in an aqueous medium and in the immobilized state, respectively. Immobilized dimers could be reconstituted to hexamers by treatment with an excess of solubilized monomers after removal of the detergent. In the presence of various phospholipids, which increased the immobilized cytochrome P-450LM2 demethylase activity and induced characteristic spectral changes, no hexamer dissociation was shown. The data obtained are thus in agreement with the suggestion that hexameric arrangement is inherent in the cytochrome P-450 when it is bound to the native membranes.

Quaternary structure of the liver microsomal cytochrome P-450.

Cytochrome P-450LM2 was isolated from rabbit liver microsomes in a form which was shown to be homogeneous in AcA-22 Ultrogel and ultracentrifugation studies. The molecular mass determined by sedimentation equilibrium roughly corresponded to hexamer composed of 56 kDa monomers. Hexamer structure of the cytochrome was directly demonstrated by electron microscopic study. In the cytochrome P-450LM2 hexamer, monomers seem to be arranged in two layers (three monomers in the layer) in such a way that each monomer occupies a position at the vertices of a triangular antiprism with a 32 point group symmetry.