Cytochrome P-450 heme moiety. The specific target in drug-induced heme alkylation.

Exogenously administered heme is incorporated into rat hepatic cytochrome P-450 in vivo (Correia, M. A., Farrell, G. C. Schmid, R. S., Ortiz de Montellano, P. R., Yost, G. S., and Mico, B. A. (1979) J. Biol. Chem. 254, 15-17). This was demonstrated in allylisopropylacetamide (AIA)-treated rats by the formation of a radioactive adduct derived from the porphyrin of the administered [3H]heme and AIA. Formation of such adducts requires catalytic participation of cytochrome P-450 in oxidative metabolism of AIA to an active species which subsequently alkylates the prosthetic heme moiety of the cytochrome. These results suggested that the exogenous heme had been incorporated prosthetically into cytochrome P-450 prior to generation of the adduct. However, the possibility remained that a minute portion of the inactivating AIA-species escaped the catalytic site of the generating hemoprotein and alkylated the nonprosthetically bound isotopic heme. To examine this critical possibility, we have employed a chemical derivative of heme which binds to the microsomal membrane. Although this heme derivative is a structurally suitable target for attack by the inactivating drug species, we found that it was unsuitable for incorporation into the prosthetic site of cytochrome P-450. The findings of this study provide irrefutable evidence that the label recovered in drug-porphyrin adducts is derived exclusively from radioactive heme incorporated prosthetically into cytochrome P-450. Drug-porphyrin adducts can therefore be used as reliable probes to follow the transfer of heme from the hepatic "free" heme pool into cytochrome P-450.