Abstract: To investigate structure-function relationships of cytochromes P450 (CYP), 3-azidiamantane was employed for photoaffinity labeling of rabbit microsomal CYP2B4. Four diamantane labeled tryptic fragments were identified by mass spectrometry and sequencing: peptide I (Leu(359)-Lys(373)), peptide II (Leu(30)-Arg(48)), peptide III (Phe(127)-Arg(140)), and peptide IV (Arg(434)-Arg(443)). Their positions were projected into CYP2B4 model structures and compared with substrate binding sites, proposed by docking of diamantane. We identified novel binding regions outside the active site of CYP2B4. One of them, defined with diamantane modified Arg(133), marks a possible entrance to the active site from the heme proximal face. In addition to crystal structures of CYP2B4 chimeras and molecular dynamics simulations, our data of photoaffinity labeling of the full CYP2B4 molecule provide further insight into functional and structural aspects of substrate binding.
Abstract: Structural analysis of the orientations of heme vinyl side chains was carried out using the published crystallographic data for different cytochromes P450. Torsional angles (tau, C(alpha)C(beta)-C(a)C(b)) show different distributions for the vinyls in positions 2 and 4. Whereas the orientation of 2-vinyls is rather restricted (tau between -120 degrees and -180 degrees ), the 4-vinyls have a much higher mobility over almost the entire conformational space. On the basis of the empirical correlation recently reported for peroxidases (M.P. Marzocchi, G. Smulevich, Relationship between heme vinyl conformation and the protein matrix in peroxidases, J. Raman Spectrosc. 34 (2003), 725-736), an attempt has been made to compare the observed vinyl orientations with the experimental frequencies of the vinyl stretching vibrational modes. The data for P450 proteins do not exactly match the peroxidase-derived function, although a qualitatively similar relationship is likely to exist. Differences between P450 forms suggest a variability in heme-region flexibility and in communication with the rest of enzyme.
Abstract: Ellipticine is an antineoplastic agent whose mode of action is based mainly on DNA intercalation, inhibition of topoisomerase II, and formation of covalent DNA adducts mediated by cytochromes P450 (P450s) and peroxidases. Here, this drug was found to induce CYP1A1 and/or 1A2 enzymes and their enzymatic activities in livers, lungs, and kidneys of rats treated (i.p.) with ellipticine. The induction is transient. In the absence of repeated administration of ellipticine, the levels and activities of the induced CYP1A decreased almost to the basal level 2 weeks after treatment. The ellipticine-mediated CYP1A induction increases the DNA adduct formation by the compound. When microsomal fractions from livers, kidneys, and lungs of rats treated with ellipticine were incubated with ellipticine, DNA adduct formation, measured by (32)P-postlabeling analysis, was up to 3.8-fold higher in incubations with microsomes from pretreated rats than with controls. The observed stimulation of DNA adduct formation by ellipticine was attributed to induction of CYP1A1 and/or 1A2-mediated increase in ellipticine oxidative activation to 13-hydroxy- and 12-hydroxyellipticine, the metabolites generating two major DNA adducts in human and rat livers. In addition to these metabolites, increased formation of the excretion products 9-hydroxy- and 7-hydroxyellipticine was also observed in microsomes of rats treated with ellipticine. Taken together, these results demonstrate for the first time that by inducing CYP1A1/2, ellipticine increases its own metabolism, leading both to an activation of this drug to reactive species-forming DNA adducts and to detoxication metabolites, thereby modulating to some extent its pharmacological and/or genotoxic potential.
Abstract: Ellipticine is an antineoplastic agent, whose mode of action is based mainly on DNA intercalation, inhibition of topoisomerase II and formation of DNA adducts mediated by cytochrome P450 (CYP). We investigated the ability of CYP enzymes in rat, rabbit and human hepatic microsomes to oxidize ellipticine and evaluated suitable animal models mimicking its oxidation in humans. Ellipticine is oxidized by microsomes of all species to 7-hydroxy-, 9-hydroxy-, 12-hydroxy-, 13-hydroxyellipticine and ellipticine N(2)-oxide. However, only rat microsomes generated the pattern of ellipticine metabolites reproducing that formed by human microsomes. While rabbit microsomes favored the production of ellipticine N(2)-oxide, human and rat microsomes predominantly formed 13-hydroxyellipticine. The species difference in expression and catalytic activities of individual CYPs in livers are the cause of these metabolic differences. Formation of 7-hydroxy- and 9-hydroxyellipticine was attributable to CYP1A in microsomes of all species. However, production of 13-hydroxy-, 12-hydroxyellipticine and ellipticine N(2)-oxide, the metabolites generating DNA adducts, was attributable to the orthologous CYPs only in rats and humans. CYP3A predominantly generates these metabolites in rat and human microsomes, while CYP2C3 activity prevails in microsomes of rabbits. The results underline the suitability of rat species as a model to evaluate human susceptibility to ellipticine.
Abstract: The series of diamondoids: adamantane, diamantane, triamantane, 2-isopropenyl-2-methyladamantane and 3-isopropenyl-3-methyldiamantane (3-IPMDIA), were employed to elucidate the molecular basis of their interaction with the active site of cytochromes P450 (CYP) of a 2B subfamily. These potent inhibitors of CYP2B enzymes were docked into the homology model of CYP2B4. Apparent dissociation constants calculated for the complexes of CYP2B4 with docked diamandoids agreed closely with the experimental data showing inhibition potency of the compounds and their binding affinity to CYP2B4. Superimposed structures of docked diamondoids mapped binding site residues. As they are mainly non-polar residues, the hydrophobicity plays the major role in the binding of diamondoids. Overlapping structure of diamondoids defined an elliptical binding cavity (5.9A inner diameter, 7.9A length) forming an angle of approximately 43 degrees with the heme plane. CYP2B specific diamondoids, namely 3-IPMDIA, showing the highest binding affinity, should be considered for a potential clinical use.
Abstract: Cytochromes P450 2E1 of human and minipig origin were examined by absorption spectroscopy under high hydrostatic pressure and by resonance Raman spectroscopy. Human enzyme tends to denature to the P420 form more easily than the minipig form; moreover, the apparent compressibility of the heme active site (as judged from a redshift of the absorption maximum with pressure) is greater than that of the minipig counterpart. Relative compactness of the minipig enzyme is also seen in the Raman spectra, where the presence of planar heme conformation was inferred from band positions characteristic of the low-spin heme with high degree of symmetry. In this respect, the CYP2E1 seems to be another example of P450 conformational heterogeneity as shown, e.g., by Davydov et al. for CYP3A4 [Biochem. Biophys. Res. Commun. 312 (2003) 121-130]. The results indicate that the flexibility of the CYP active site is likely one of its basic structural characteristics.
Abstract: Aristolochic acid (AA), a naturally occurring nephrotoxin and carcinogen, has been associated with the development of urothelial cancer in humans. Using the 32P-postlabeling assay we showed that AAI is activated by human recombinant cytochrome P450 (CYP) 1A1, CYP1A2 and NADPH:CYP reductase to species generating DNA adduct patterns reproducing those found in renal tissues from humans exposed to AA. 7-(Deoxyadenosin-N6-yl)aristolactam I, 7-(deoxyguanosin-N2-yl)aristolactam I and 7-(deoxyadenosin-N6-yl)aristolactam II were identified as AA-DNA adducts formed from AAI by the enzymes. The formation of these AA-derived DNA adducts indicates that all the human enzymes reduce the nitro group of AAI to the putative reactive cyclic nitrenium ion responsible for adduct formation. The concentrations of AAI required for its half-maximum DNA binding were 38, 65 and 126 microM AAI for reductive activation by human CYP1A2, CYP1A1 and NADPH:CYP reductase, respectively. CYP1A1 and 1A2 homology modeling followed by docking of AAI to the CYP1A1 and 1A2 active centers was utilized to explain the potential of these enzymes to reduce AAI. Models of human CYP1A1 and 1A2 were constructed on the basis of the crystallographic structure of truncated mammalian CYP enzymes, CYP2B4, 2C5, 2C8, 2C9 and 3A4. The in silico docking of AAI to the active sites of CYP1A1 and 1A2 indicates that AAI binds as an axial ligand of the heme iron and that the nitro group of AAI is in close vicinity to the heme iron of CYP1A2 in an orientation allowing the efficient reduction of this group observed experimentally. The orientation of AAI in the active centre of CYP1A1 however causes an interaction of the heme iron with both the nitro- and the carboxylic groups of AAI. This observation explains the lower reductive potential of CYP1A1 for AAI than CYP1A2, detected experimentally.
Abstract: 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 N(2)-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 N(2)-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 N(2)-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 N(2)-oxide.
Abstract: Spectroscopic methods reveal differences in flexibility and stability of P450 forms. Among microsomal P450s, the most flexible active site has been found in the CYP3A4 enzyme as it is compressible and the heme vinyl side chains may adopt two different conformations. On the other hand, active site of this enzyme denatures quite easily upon hydrostatic pressure. The most rigid active site able to withstand the effect of high pressure has CYP1A2. The bacterial CYP102 (BM3) flavocytochrome has also a rather stable, but flexible active site. The differences between CYP3A4 and CYP1A2 active sites apparently reflect their ability to bind various substrates: whereas the CYP3A4 binds a vast variety of structures, the CYP1A2 preferentially binds planar, aromatic structures and its substrate specificity is relatively narrow.
Abstract: Cytochromes P450 CYP102 A1, 1A2, and 3A4, all belonging to the class II type of P450 enzymes, were studied by resonance Raman spectroscopy. Spectra were measured for the oxidized substrate-free, oxidized substrate-bound, and reduced forms of each of these P450s. The analysis of the resonance Raman spectra indicates that the individual isoforms differ with respect to orientation and conformations of the heme side chains, whereas the overall porphyrin geometry is essentially the same. In the oxidized state, the vinyl groups exhibit both a coplanar and an out-of-plane orientation with respect to the heme, albeit with different relative propensities in the various isoforms. In the reduced state, both vinyl groups are forced into a coplanar orientation. In addition to the differences in behavior of the vinyl groups, the redox-linked spectral changes also include the bending mode of the propionate side chains. The spectral differences associated with the porphyrin substituents are likely to reflect subtle conformational differences in the heme pocket of various P450 isoforms which may constitute the structural basis for the known variability of their functions.
Abstract: The flexibility of the structure and compressibility of the respective active site of cytochromes P450 3A4 (CYP3A4) and BM-3 (CYP102) were studied using absorption spectroscopy in the ultraviolet and visual regions. Conformational changes in the overall protein structures of both CYP3A4 and CYP102 due to the effects of temperature and pressure are reversible. However, the enzymes differ in the properties of their active sites. The CYP3A4 enzyme denatures to the inactive P420 form relatively easy, at 3000 bar over half is converted to P420. The compressibility of its active site is lower than that of CYP102 and is greater with the substrate bound, which is in line with the observed lack of a stabilizing effect of the substrate on its conformation under pressure. In contrast, CYP102, although having the most compressible active site among the P450s, possesses a structure that does not denature easily to the inactive (P420) form under pressure. In this respect, it resembles the P450 isolated from acidothermophilic archaebacteria [McLean, M.A., Maves, S.A., Weiss, K.E., Krepich, S. & Sligar, S.G. (1998) Biochem. Biophys. Res. Commun. 252, 166-172].
Abstract: 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.
Abstract: The resonance Raman spectra of CYP102 holoenzyme and of the CYP102 heme domain in the reduced state have been obtained for the first time. Spectra of the oxidized heme domain have also been measured. Whereas the spectra of the isolated heme domain are similar to those obtained for other hexacoordinated low-spin P450s, the holoenzyme spectra exhibited unexpected features. The most plausible explanation is that they reflect an electron transfer to the heme from photoreduced flavins. The results obtained for both the oxidized and reduced heme domain bring additional support to the use of CYP102 as a model for microsomal mammalian P450 enzymes, showing that the heme moiety in CYP102 has similar properties to the hemes in microsomal P450s.
Abstract: Fluorescence of eight tryptophan residues in cytochrome P-450scc with bound endogenous cholesterol could be fitted with a two component model: a single exponential and a "top-hat" distribution of lifetimes as the second component. The short-lived component (tau 1 about 700 ps) does not change significantly upon binding of substrate (22R-hydroxycholesterol). The parameters of the long-lived component (central lifetime tau m about 3.4 ns) change upon binding of carbon monoxide and substrate. 22R-hydroxycholesterol binding broadens the distribution of the long-lived component; that is the heterogeneity of the Trp environment is increased when this substrate displaces the endogenous cholesterol.
Abstract: Properties of the single tryptophan residue in rat liver microsomal phenobarbital-inducible cytochrome P-450e (P450IIB2) were studied by the nanosecond time-resolved fluorometry. The tryptophan fluorescence decay time was found to be 3.6 ns and it was not affected by the addition of substrate (perhydrophenanthrene). This result strongly indicates that the tryptophan residue is not a part of the substrate-binding site.
Abstract: The secondary structure of rabbit liver microsomal cytochrome P-450 LM2, rat liver microsomal cytochromes P-450b and P-450e (phenobarbital-inducible), and rat liver microsomal cytochromes P-450c, P-450d (3-methylcholanthrene-inducible) was predicted by a combination of methods (i) identifying the transmembrane parts of integral membrane proteins, and (ii) statistically predicting the secondary structure of globular proteins. The results are similar for all phenobarbital-inducible enzymes and make it possible to construct two structural models with seven or four transmembrane alpha-helices. The cytochromes of the second group obviously form a second structural family with four membrane-spanning alpha-helices. In both cases, a large ectodomain with several consecutive alpha-helices, which may provide the heme-binding pocket, is exposed out of the membrane.
