Homology models of the active site of cytochrome P450 2D6 (CYP2D6) have identified phenylalanine 481 (Phe(481)) as a putative ligand-binding residue, its aromatic side chain being potentially capable of participating in pi-pi interactions with the benzene ring of ligands. We have tested this hypothesis by replacing Phe(481) with tyrosine (Phe(481)-->Tyr), a conservative substitution, and with leucine (Phe(481)-->Leu) or glycine (Phe(481)-->Gly), two non-aromatic residues, and have compared the properties of the wild-type and mutant enzymes in microsomes prepared from yeast cells expressing the appropriate cDNA-derived protein. The Phe(481)-->Tyr substitution did not alter the kinetics [K(m) (microM) and V(max) (pmol/min per pmol) respectively] of oxidation of S-metoprolol (27; 4.60), debrisoquine (46; 2.46) or dextromethorphan (2; 8.43) relative to the respective wild-type values [S-metoprolol (26; 3.48), debrisoquine (51; 3.20) and dextromethorphan (2; 8.16)]. The binding capacities [K(s) (microM)] of a range of CYP2D6 ligands to the Phe(481)-->Tyr enzyme (S-metoprolol, 22.8; debrisoquine, 12.5; dextromethorphan, 2.3; quinidine, 0.13) were also similar to those for the wild-type enzyme (S-metoprolol, 10.9; debrisoquine, 8.9; dextromethorphan, 3.1; quinidine, 0.10). In contrast, the Phe(481)-->Leu and Phe(481)-->Gly substitutions increased significantly (3-16-fold) the K(m) values of oxidation of the three substrates [S-metoprolol (120-124 microM), debrisoquine (152-184 microM) and dextromethorphan (20-31 microM)]. Similarly, the K(s) values of the ligands to Phe(481)-->Leu and Phe(481)-->Gly mutants were also increased 3 to 10-fold (S-metoprolol, 33.2-41.9 microM; debrisoquine, 85-90 microM; dextromethorphan, 15.7-18.8 microM; quinidine 0.35-0.53 microM). However, contrary to a recent proposal that Phe(481) has the dominant role in the binding of substrates that undergo CYP2D6-mediated N-dealkylation routes of metabolism, the Phe(481)-->Gly substitution did not substantially decrease the capacity of the enzyme to N-deisopropylate metoprolol (wild-type, 1.12 pmol/min per pmol of P450; Phe(481)-->Gly, 0.71), whereas an Asp(301)-->Gly substitution decreased the N-dealkylation reaction by 95% of the wild-type rate. Overall, our results are consistent with the proposal that Phe(481) is a ligand-binding residue in the active site of CYP2D6 and that the residue interacts with ligands via a pi-pi interaction between its phenyl ring and the aromatic moiety of the ligand. However, the relative importance of Phe(481) in binding is ligand-dependent; furthermore, its importance is secondary to that of Asp(301). Finally, contrary to predictions of a recent homology model, Phe(481) does not seem to have a primary role in CYP2D6-mediated N-dealkylation.