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, FeO 2 − ) or Compound I (perferryl oxygen, FeO 3+ ) reacting with either the aldehyde or its hydrate, respectively. Analysis of 18 O incorporation into formic acid from 18 O 2 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 FeO 2 − 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 H 2 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.