Oxygen-18 Labeling Reveals a Mixed Fe-O Mechanism in the Last Step of Cytochrome P450 51 Sterol 14α-Demethylation

The 14α-demethylation step is critical in eukaryotic sterol biosynthesis, catalyzed by cytochrome P450 (P450) Family 51 enzymes, for example, with lanosterol in mammals. This conserved three-step reaction terminates in a C-C cleavage step that generates formic acid, the nature of which has been controversial. Proposed mechanisms involve roles of P450 Compound 0 (ferric peroxide anion, FeO₂ ^- ) or Compound I (perferryl oxygen, FeO³⁺ ) reacting with either the aldehyde or its hydrate, respectively. Analysis of ¹⁸ O incorporation into formic acid from ¹⁸ O₂ provides a means of distinguishing the two mechanisms. Human P450 51A1 incorporated 88 % ¹⁸ O (one atom) into formic acid, consistent with a major but not exclusive FeO₂ ^- mechanism. Two P450 51 orthologs from amoeba and yeast showed similar results, while two orthologs from pathogenic trypanosomes showed roughly equal contributions of both mechanisms. An X-ray crystal structure of the human enzyme showed the aldehyde oxygen atom 3.5 Å away from the heme iron atom. Experiments with human P450 51A1 and H₂ ¹⁸ O yielded primarily one ¹⁸ O atom but 14 % of the formic acid product with two ¹⁸ O atoms, indicative of a minor contribution of a Compound I mechanism. LC-MS evidence for a Compound 0-derived Baeyer-Villiger reaction product (a 14α-formyl ester) was also found.