2-Ethynylnaphthalene (2EN) is a mechanism-based inactivator of P450 2B4 that covalently modifies an amino acid in the peptide Glu273-Met314 with a 2-naphthylacetyl group [Roberts et al. (1994) Biochemistry 33, 3766-3771]. Truncated 2B4 lacking amino acids 2-27, 2B4 (Delta2-27), was expressed in Escherichia coli, purified, and found to catalyze the oxidation of 2EN to 2-naphthylacetic acid (2NA). The metabolism of 2EN resulted in the inactivation and covalent modification of the protein moiety as we have previously reported with P450 2B4 purified from the livers of phenobarbital-induced rabbits. The rate constants of inactivation of the O-deethylation activity of 7-ethoxy-4-trifluoromethylcoumarin (EFC) were 0.15 +/- 0.01 and 0.20 +/- 0.05 min-1 for the protein purified from rabbit liver and 2B4 (Delta2-27), respectively. A protein in which threonine 302 was replaced with alanine, P450 2B4 (Delta2-27, T302A), was inactivated by 2EN with a much slower rate constant (0.05 +/- 0.01 min-1) and formed 1.8-fold more 2NA as compared to P450 2B4 (Delta2-27) over a 10-min incubation. When the formation of 2NA was supported by cumene hydroperoxide, 2B4 (Delta2-27, T302A) formed 30% less product than 2B4 (Delta2-27) over a 5-min incubation. After incubation with [3H]2EN and NADPH, P450 2B4 (Delta2-27) had significant radioactivity associated with the P450 in an NADPH-dependent manner when the incubation mixture was analyzed by SDS-PAGE followed by autoradiography and 10-fold more radioactivity associated with the P450 as compared to P450 2B4 (Delta2-27, T302A) when analyzed by reverse-phase HPLC. Thus, threonine 302 is not required by 2B4 for oxidation of 2EN or EFC but appears to play an important role in the inactivation of P450 2B4 by 2EN and the covalent labeling of the P450 protein by 2EN.