Cytochrome P-450 hemoproteins oxidize chemically diverse endogenous and exogenous substrates (drugs, toxins). Although their biosynthetic regulation is somewhat understood, little is known about their degradation except that the apoprotein and heme moieties turn over asynchronously. But the trigger and the cellular loci for these events remain to be identified. we have found that administration of the diuretic spironolactone, or DDEP, a structural analog of dihydropyridine Ca2+ antagonists, results in rapid structural and functional loss of hepatic P-450p isozymes in rats. Such P- 450p loss appears to proceed via a novel mechanism-based """"""""suicide"""""""" inactivation which alkylate the apoprotein at the chemical structure of the heme=protein adducts are unknown but appear to incur one- (but not two-) electron oxidation of DDEP. DDEP-mediated prosthetic heme alkylation of the apocytochrome also occurs in vivo and is associated with accelerated loss of immunochemically detectable apocytochrome P-450p. Intriguingly, low but detectable levels of heme-alkylated apoprotein are also observed in untreated rats, a feature enhanced by P-450p induction. Together these findings raise the provocative possibility that heme alkylation of apo-P- 450p by structurally modifying the protein may function as a stimulus for its proteolytic removal. Indeed its endogenous occurrence albeit at lower rates suggests that it might in fact represent a normal pathway for its turnover. Alternatively, since le- oxidation of DDEP by P-450 generates a highly reactive """"""""compound II""""""""-like [Fe(IV)=O/Fe(III)OH.] species, the possibility of apocytochrome P-450 cooxidation at its active site in addition to oxidation self-destruction of the prosthetic heme also must be considered. Studies are proposed to determine the normal extent of apo-P- 450 heme alkylation to examine oxidative modification of the apoprotein, to structurally characterize the apoprotein-alkylating heme species, to determine the proteolytic susceptibility of such heme-alkylated proteins, and, finally, to identify the intracellular loci/proteases of such an event. We believe these studies are important not only because they address a basic but ill-understood aspect of P-450 biology but also because the isozyme in question is homologous to that induced in humans by various commonly prescribed drugs (gluco-corticoids, macrolide antibiotics, and anticonvulsants). Also, elucidation of P-450p degradation may shed light on the degradation of other cellular hemoprotein oxidases.
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