The porphyrinogenic agent 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1, 4-dihydroxy pyridine (DDEP) is known to destroy hepatic cytochrome P450 isoforms (2C11, 2C6 and 3A1) in a mechanism-based inactivation process. Horseradish peroxidase (HRP) was used as a model system for the DDEP mediated inactivation of these isoforms. Two major metabolites of DDEP, formed by K3Fe(CN)6 and by HRP and hydrogen peroxide, were 3,5-dicarbethoxy-2,6-dimethyl-pyridine (DP) and 3,5-dicarbethoxy-2, 6-dimethyl-4-ethylpyridine (EDP). In both of these systems, EDP is the predominant metabolite at acidic and neutral pH values whereas DP is the major product under slightly alkaline conditions. Evidently, the relative rates of formation these metabolites is controlled solely by the pH of media and not by the enzyme. The inactivation of HRP by ethylhydrazine (EH) was studied as an alternative mechanism for the formation of ethyl radical-mediated alterations of heme. The observed pH profile of heme loss by EH exactly corresponds to that of DDEP. While HRP was not inactivated under acidic conditions, it was inactivated with the formation of altered heme products at pH 8. An altered heme product of HRP by DDEP was isolated by HPLC. FAB mass spectra and NMR showed that the altered heme contained a 2-hydroxy ethyl group attached to the omega-meso carbon of heme. The semiempirical quantum chemical method, AM1, as provided in MOPC 6.0 was used to calculate the heats of reaction in order to predict possible reaction pathways. The results of these calculations indicated that DDEP first undergoes a one-electron oxidation to form a N radical cation, but that the fate of the radical cation depends on the presence of acidic and basic groups in the active site of the enzyme. The predominant formation of DP in P450 2C11 and 2C6 catalyzed reactions indicates that the active site of these isozymes are very lipophilic and basic, which allows the N radical cation to deprotonate rapidly and then to disproportionate into DP and the ethyl radical. On the other hand, the predominate formation of EDP by P450 3A indicates that its active site is polar and acidic, which facilitates the oxidation of the N radical cation to EDP.
