Jiri Hudecek

The Anticancer Drug Ellipticine Forms Covalent DNA Adducts, Mediated by Human Cytochromes P450, through Metabolism to 13-Hydroxyellipticine and Ellipticine N2-Oxide

Ellipticine is an antineoplastic agent, the mode of action of which is considered to be based on DNA intercalation and inhibition of topoisomerase II. We found that ellipticine also forms the cytochrome P450 (CYP)-mediated covalent DNA adducts. We now identified the ellipticine metabolites formed by human CYPs and elucidated the metabolites responsible for DNA binding. The 7-hydroxyellipticine, 9-hydroxyellipticine, 12-hydroxyellipticine, 13-hydroxyellipticine, and ellipticine N2-oxide are generated by hepatic microsomes from eight human donors. The role of specific CYPs in the oxidation of ellipticine and the role of the ellipticine metabolites in the formation of DNA adducts were investigated by correlating the levels of metabolites formed in each microsomal sample with CYP activities and with the levels of the ellipticine-derived deoxyguanosine adducts in DNA. On the basis of this analysis, formation of 9-hydroxyellipticine and 7-hydroxyellipticine was attributable to CYP1A1/2, whereas production of 13-hydroxyellipticine and ellipticine N2-oxide, the metabolites responsible for formation of two major DNA adducts, was attributable to CYP3A4. Using recombinant human enzymes, oxidation of ellipticine to 9-hydroxyellipticine and 7-hydroxyellipticine by CYP1A1/2 and to 13-hydroxyellipticine and N2-oxide by CYP3A4 was corroborated. Homologue modeling and docking of ellipticine to the CYP3A4 active center was used to explain the predominance of ellipticine oxidation by CYP3A4 to 13-hydroxyellipticine and N2-oxide.

Hydrophobic/hydrophilic interactions of cytochrome c with functionalized self-assembled monolayers on silicon

Abstract Cytochrome c (C c , horse heart) has been adsorbed onto self-assembled monolayers (SAM) on silicon single crystal substrates. Layer thickness was determined using ellipsometry and atomic force microscopy in the DFM tapping mode. Both hydrophilic (COOH containing SAM) and hydrophobic self-assembled monolayers were used. The protein layers were found to consist of adsorbed 2-dimensional islands. Concentration, exposure time and the defect-density of the self-assembled monolayer substrates determined the wetting properties of the resulting layer, indicating that the surface orientation of the protein is driven by the interaction with the substrate. On well ordered self-assembled monolayers, the protein layer thicknesses were 1.76 nm for charged surfaces and 2.3 nm for hydrophobic surfaces. Self-assembled monolayers of a lower density resulted in a prevalence of C c islands of 3.2 nm thickness for both cases.

Structural changes in cytochrome c oxidase induced by cytochrome c binding. A resonance Raman study

Electrostatically stabilized complexes of fully oxidized cytochrome c oxidase from Paracoccus denitrificans and horse heart cytochrome c were studied by resonance Raman spectroscopy. The experiments were carried out with the wild-type oxidase and a variant in which a negatively charged amino acid in the binding domain (D257) is replaced by an asparagine. It is shown that cytochrome c induces structural changes at heme a and heme a(3) which are reminiscent to those found in mammalian cytochrome c oxidase-cytochrome c complex. The spectral changes are attributed to subtle changes in the heme-protein interactions implying that there is a structural communication from the binding domain even to the remote catalytic center. Only for the heme a modes minor spectral differences were found in the response of the wild-type and the D257N variant oxidase upon cytochrome c binding indicating that electrostatic interactions of aspartate 257 are not crucial for the perturbation of the catalytic site structure in the complex. On the other hand, in none of the complexes, structural changes were detected in the bound cytochrome c. These findings are in contrast to previous results obtained with beef heart cytochrome c oxidase which triggers the formation of a new conformational state of cytochrome c assumed to be involved in the biological electron transfer process.

Physics and Biochemistry: How Much They Can Have in Common

The first part of this contribution deals with the relationships between, physics and biology, and with contents of biophysical and biochemical research. In the second part, some biochemical problems of potential interest to the physicists are treated in more detail (energetic metabolisms, structure of globular proteins).