Metabolic Activation oftrans-4-Hydroxy-2-nonenal, a Toxic Product of Membrane Lipid Peroxidation and Inhibitor of P450 Cytochromes

Chung-Liang Kuo, Alfin D. N. Vaz, Minor J. Coon
1997 Journal of Biological Chemistry  
Lipid peroxidation in biological membranes is known to yield reactive aldehydes, of which trans-4-hydroxy-2nonenal (HNE) is particularly cytotoxic. This laboratory previously reported that purified liver microsomal P450 cytochromes are directly inactivated to varying extents by HNE. We have now found a mechanism-based reaction in which P450s are inactivated by HNE in the presence of molecular oxygen, NADPH, and NADPH-cytochrome P450 reductase. The sensitivity of the various isozymes in the two
more » ... athways is different as follows: P450 2B4 and the orthologous 2B1 are inactivated to the greatest extent and 2C3, 1A2, 2E1, and 1A1 to a somewhat lesser extent by the pathway in which HNE undergoes metabolic activation. In contrast, 2B4 and 2B1 are insensitive to direct inactivation, and the reductase is unaffected by HNE by either route. Recent studies on the catalytic activities of the T302A mutant of P450 2B4 have shown that the rate of oxidation of a variety of xenobiotic aldehydes to carboxylic acids is decreased, but the rates of aldehyde deformylation and mechanismbased inactivation of the cytochrome are stimulated over those of the wild-type enzyme (Raner, G. M., Vaz, A. D. N., and Coon, M. J. (1997) Biochemistry 36, 4895-4902). Inactivation by those aldehydes apparently occurs by homolytic cleavage of a peroxyhemiacetal intermediate to yield formate and an alkyl radical that reacts with the heme. In sharp contrast, the rate of mechanismbased inactivation by HNE is decreased with the T302A mutant relative to that of the wild-type P450 2B4, and mass spectral analysis of the heme adduct formed shows that deformylation does not occur. We therefore propose that the metabolic activation of HNE involves formation of an acyl carbon radical that leads to the carboxylic acid or alternatively reacts with the heme. The toxicity of lipid peroxidation products generated in biological membranes is widely attributed to reactive aldehydes, including 2-alkenals and 4-hydroxyalkenals (1). Of these, trans-4-hydroxy-2-nonenal (HNE) 1 is produced in relatively large amounts and is particularly deleterious in that it is cytotoxic (2), lyses erythrocytes (3) , and inhibits DNA and protein synthesis (4, 5). Stadtman and colleagues (6 -8) have described the inactivation of purified cytosolic enzymes by HNE and have shown the involvement of protein sulfhydryl, amino, and imidazole groups. Since the first target of the toxic products of lipid peroxidation would be expected to be membranous enzymes, this laboratory recently undertook a study of the sensitivity of hepatic microsomal P450 (see Ref. 10 for updated P450 nomenclature) cytochromes to HNE (9). The six purified isozymes that were examined were found to differ in the effect of this agent at various concentrations, with P450s 1A1, 2E1, and 1A2 being most readily inactivated. The present paper is concerned with a newly discovered reaction in which HNE undergoes metabolic activation by cytochrome P450 in a reconstituted oxygenating system containing molecular oxygen, NADPH, and the reductase. All of the P450 isozymes examined are inactivated in this mechanismbased reaction, but the pattern is strikingly different from that seen in the earlier studies on the direct effect of HNE, that is in the absence of NADPH and reductase. For example, P450 2B4, the phenobarbital-inducible isozyme (11), is unaffected by direct exposure to HNE but is the most sensitive of all of the cytochromes examined to metabolically activated HNE. This laboratory has been interested in the deformylation of aldehydes as a model for the final step in the aromatasecatalyzed reaction in which androgens are converted to estrogens (12) and has shown that similar oxidative cleavages occur with purified liver microsomal P450s. The reactions include the conversion of cyclohexane carboxaldehyde to cyclohexene (13), of a series of low molecular weight branched chain aldehydes to the n Ϫ 1 alkenes, and of citronellal to 2,6-dimethyl-1,5-heptadiene (14), and of 3-oxodecalin-4-ene-10-carboxaldehyde, a bicyclic steroid analog, to 3-hydroxy-6,7,8,9-tetrahydronaphthalene (15). Our recent investigation on the effect of site-directed mutagenesis of threonine 302 to alanine on the activities of recombinant P450 2B4 (16) drew on earlier evidence from others that the corresponding mutation in bacterial P450cam interferes with the activation of dioxygen to the oxenoid species, apparently by disrupting proton delivery to the active site (17, 18) . Of particular interest, the deformylation of cyclohexane carboxaldehyde by the mutant 2B4 P450 to produce cyclohexene is greatly increased, which along with other evidence led us to conclude that the iron-peroxy species, rather than the ironoxene species, is the direct oxygen donor. More recently, we have examined the mechanism by which a series of xenobiotic aldehydes bring about the mechanismbased inactivation of P450 2B4 (19). The results obtained with the T302A mutant of P450 2B4, along with isotope effects and observations on the influence of aldehyde structure, indicated that an alkyl free radical produced upon decomposition of the
doi:10.1074/jbc.272.36.22611 pmid:9278417 fatcat:5nc3blsnrffuzlizcwjb4j465m