Comparisons of hydroperoxide isomerase and monooxygenase activities of cytochrome P450 for conversions of allylic hydroperoxides and alcohols to epoxyalcohols and diols: probing substrate reorientation in the active site.

Ferric cytochrome P450 (P450) catalyzes intramolecular rearrangements of hydroperoxides to diols by heterolytic scission of the O-O bond and insertion of the terminal oxygen atom into the resulting alcohol. The goals of this work were to further characterize the regio- and stereochemistry of P450 isomerase activity using allylic hydroperoxides and to compare these reactions with NADPH-supported monooxygenations of the corresponding alcohols. Microsomes from phenobarbital-treated rats or purified P450 2B1 catalyzed the conversions of several peroxyquinols, including 2-tert-butyl-4-hydroperoxy-4-methylcyclohexa-2,5-dien-1-one (BMPOOH) and its 2,6-dimethyl derivative (TMPOOH), to diols and to alpha,beta-epoxyquinols by predominant (>93%) cis addition of oxygen to the least hindered double bond. Monooxygenation of the 4-hydroxy analogues (quinols) yielded identical cis-epoxyquinols, and both isomerization and monooxygenation reactions exhibited similar enantioselectivities (32-74% enantiomer excess). Regioselectivities were similar for BMPOOH and BMPOH (epoxyquinol:diol ratios of 5.8-7.6), but quite different for TMPOOH and TMPOH (ratios of 0.6 and 6.4, respectively). Bulky peroxyquinols and quinols derived from the A-rings of 17beta-estradiol and estrone were utilized to further compare these reactions. Both estradiol derivatives underwent approximately equal amounts of 6beta-hydroxylation and 1,2-epoxidation. The estrone derivatives also underwent 6beta-hydroxylation, but only estrone quinol yielded a second product consistent with hydroxylation at position 16. The results support several conclusions. (i) Allylic hydroperoxides may be isomerized to alpha,beta-epoxyalcohols by a heterolytic O-O cleavage mechanism with high stereoselectivity. (ii) Hydroperoxide isomerization is an efficient process relative to monooxygenation. (iii) Isomerase substrates remain in proximity to the P450 oxoferryl intermediate and are rapidly captured by the oxidant. Monooxygenase substrates, on the other hand, may bind to ferric P450 in multiple orientations and undergo more extensive substrate reorientation prior to oxidative attack.

Understanding the cellular uptake of phosphopeptides.

Phosphopeptide-cellular uptake has been studied with a unique combination of tools designed to quantitate this phenomena and to understand properties that contribute to transmembrane penetration. High-affinity src-homology domain (SH2) hexapeptides for the phosphatidyl inositol 3-kinase system were used to judge cell penetration using red blood cells--a model system for the study of transmembrane cellular uptake. Hexapeptides without phosphate groups and devoid of charged residues poorly entered cells. N-terminal modification with bulky hydrophobic groups enhanced partitioning into octanol, an index of hydrophobicity, and allowed certain non-phosphorylated peptides to pass into red cells. However, tyrosine phosphorylation of hexapeptides markedly decreased octanol-water partitioning and completely eliminated cellular uptake. Inclusion of ion-pairing agents that masked the phosphate hydrophilic character enabled partitioning of phosphopeptides into octanol and achieved cellular uptake. This effect was demonstrated using fluorescent derivatives of phosphopeptides and CV1 cells in culture. The results validate the concept of facilitating cell entry by charge masking and open the way to future refinements of this principle. Various penetration techniques are compared and discussed in the context of maximizing cellular viability.

Interactions of peroxyquinols with cytochromes P450 2B1, 3A1, and 3A5: influence of the apoprotein on heterolytic versus homolytic O-O bond cleavage.

In studies of the mechanisms involved in oxygen activation by cytochromes P450 (P450s), organic hydroperoxides are frequently used to model the putative peroxyferrous complex formed immediately prior to oxygen-oxygen bond cleavage during the reaction cycle of this monooxygenase. Heterolysis of the O-O bond by ferric P450 generates the catalytically competent oxo-ferryl intermediate analogous to peroxidase Compound I, and homolysis produces the Compound II analog capable only of one-electron oxidations. As P450s have been shown to catalyze both modes of O-O bond scission, the present investigation was focused on the influence of the apoprotein on the relative contributions of these competing processes. Liver microsomes from rats treated with the P450-inducing agents phenobarbital, beta-naphthoflavone, and dexamethasone, as well as purified P450s 2B1, 3A1, and 3A5 were incubated with 2,6-di-tert-butyl-4-hydroperoxy-4-methyl-2, 5-cyclohexadienone (BHTOOH). Ratios of heterolysis to homolysis were determined by analyzing the products derived from this hydroperoxide. The data demonstrate that BHTOOH is cleaved with a ratio of approximately 1.0 with all of the liver microsomal or purified P450s investigated, except with liver microsomes from dexamethasone-treated rats or with P450s 3A1 and 3A5. In these cases, heterolysis predominated over homolysis by factors of 2.5 to 4.0. On the other hand, microsomes rich in P450 2B1 catalyzed predominantly heterolysis with analogs of BHTOOH containing smaller alkyl substituents. The data are consistent with a requirement for solvent access to the peroxyferrous complex and general acid catalysis of heterolytic O-O bond cleavage by water.

Metabolic activation of butylated hydroxytoluene by mouse bronchiolar Clara cells.

Metabolism of BHT (2,6-di-tert-butyl-4-methylphenol) is requisite for its pneumotoxic activities. Previous evidence using microsomal preparations from livers and lungs of mice indicated that cytochrome P450-catalyzed hydroxylation of a tertbutyl group to form 6-tert-butyl-2-(hydroxy-tert-butyl)-4-methylphenol (BHTOH) is the first step in the bioactivation of this compound. Subsequent oxidation of BHTOH produces the quinone methide 6-tert-butyl-2-(hydroxy-tert-butyl)-4-methylene-2,5-cyclohexadienone (BHTOH-QM), and this highly reactive electrophile may be directly responsible for the pulmonary effects of BHT. The present study assessed the ability of intact bronchiolar Clara cells isolated from mice, a major site of pulmonary xenobiotic metabolism, to convert BHT to BHTOH-QM. The data demonstrate that BHTOH is, in fact, the principal oxidation product in these cells, and that a substantial portion of this metabolite is oxidized further to the quinone methide. BHTOH was found to be considerably more toxic to Clara cells than BHT, and both toxicity and metabolism were simultaneously depressed by the cytochrome P450 inhibitor SKF 525-A. These results strongly support the hypothesis that BHTOH-QM is the active metabolite that generates acute pneumotoxicity and modulates lung tumor formation.

Analysis of trimethylsilylperoxy derivatives of thermally labile hydroperoxides by gas chromatography-mass spectrometry.

Qualitative and quantitative determinations of hydroperoxides are of central importance in many areas of biochemical research, but analysis of intact hydroperoxides by capillary gas chromatography-mass spectrometry (GC-MS) generally has not been possible due to the thermal instability of these compounds. It is demonstrated here that structurally diverse hydroperoxides can be converted to trimethylsilyl (TMS) peroxides and analyzed by GC-MS without significant thermal decomposition. The hydroperoxides investigated in this report include cumyl hydroperoxide, 2-phenylethylhydroperoxide, 13-hydroperoxy-octadeca-9,11-dienoic acid, 2,6-di-tert-butyl-4-hydroperoxy-4-methylcyclohexadienone (BHTOOH), and 2,4,6-trimeth-yl-4-hydroperoxycyclohexadienone (TMPOOH). A representative peracid, 3-chloroperoxybenzoic acid, was successfully analyzed as its TMS peroxyester. Chromatographic and mass spectral characteristics of the TMS peroxides and their corresponding TMS ethers were obtained and compared with thermospray HPLC-MS data for underivatized hydroperoxides. The utility of the GC-MS assay for quantitating low levels of hydroperoxides in biological matrices was demonstrated by measuring BHTOOH and TMPOOH formed during the NADPH-dependent oxidation of alkylated phenols by rat liver microsomes. Overall, the results demonstrate that the advantages of GC-MS analysis (i.e., high chromatographic resolution, quantitative precision, high sensitivity, and the acquisition of structural information) can be applied successfully to intact hydroperoxides following trimethylsilylation.