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Polyhalogenated compounds, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, are associated with toxic Uroporphyria and cause alleviation of jaundice in the Gunn rat. These effects have been attributed to a microsomal oxidation of uroporphyrinogen and bilirubin for which supportive evidence has been obtained in vitro. CYP1A1 required planar polyhalogenated biphenyls for these oxidative reactions, while CYP1A2 was capable of oxidation in their absence.We have now used rat CYP1A1 and confirmed with the pure enzyme that increased bilirubin oxidation was caused by the addition of 3,4,3″,4″-tetrachlorobiphenyl. CYP1A2 was more active than CYP1A1 at oxidizing bilirubin in presence of NADPH alone and reacted to addition of 3,4,3″,4″-tetrachlorobiphenyl with a depression rather than a stimulation of bilirubin oxidation.We have also tested a bacterial enzyme, CYP102. Dodecanoic acid and its polyhalogenated analogue (perfluorododecanoic acid) both stimulated NADPH oxidation by CYP102, but only the perfluoro analogue stimulated markedly bilirubin oxidation. The analogue exhibited much greater potency than the normal substrate in stimulating NADPH and bilirubin oxidation and also showed greater affinity for CYP102, as measured by the binding constant, Ks. The molar stoichiometry ratio between NADPH and O2 consumption was 1 in the case of the substrate, but approximated 2 with the perfluoro analogue. We conclude that halogenated substrate analogues can interact with different CYPs to increase production of oxidative species, probably by an uncoupling mechanism. A role of the ferryl-oxygen intermediate is suggested in the oxidation of biologically important molecules, with possible implications for the therapy of jaundice and for toxic oxidative reactions, such as uroporphyria and cancer.