CYP73A1 is a typical plant P450 in terms of its function and primary sequence. The enzyme catalyzes the 4-hydroxylation of trans-cinnamic acid, the first oxidative step in the phenylpropanoid pathway. Its primary protein sequence exhibits some particular landmarks which are characteristic of plant P450 enzymes. The most interesting is a proline residue (Pro448), very unusual in animal P450s, just C-terminal to the invariant heme-binding cysteine. To determine the role of this proline, we substituted it with valine, isoleucine, or phenylalanine, residues found in animal P450s, using site-directed mutagenesis. Expression of the wild type and mutants in yeast indicated that replacement of Pro448 led to disruption of the heme-protein interaction, loss of catalytic activity, and either impaired expression or destabilization of the apoprotein. Pro448 is thus essential for the correct insertion of heme in the apoprotein. Another typical feature of CYP73A proteins is the presence of an alanine-alanine motif (Ala306-Ala307) on the presumed N-terminal edge of the cleft in the central part of the I helix. This cleft faces the iron on the distal side of the heme and is proposed to be essential for catalysis. Substitution of each or both Ala306 and Ala307 residues with glycines showed that they are critical for the stability of the protein and influence the positioning of the substrate in the active site. Results are discussed with reference to the structural X-ray data that are available for bacterial P450 proteins.