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This conserved three\u2010step reaction terminates in a C\u2212C 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<jats:sub>2<\/jats:sub><jats:sup>\u2212<\/jats:sup>) or Compound I (perferryl oxygen, FeO<jats:sup>3+<\/jats:sup>) reacting with either the aldehyde or its hydrate, respectively. Analysis of <jats:sup>18<\/jats:sup>O incorporation into formic acid from <jats:sup>18<\/jats:sup>O<jats:sub>2<\/jats:sub> provides a means of distinguishing the two mechanisms. Human P450 51A1 incorporated 88\u2009% <jats:sup>18<\/jats:sup>O (one atom) into formic acid, consistent with a major but not exclusive FeO<jats:sub>2<\/jats:sub><jats:sup>\u2212<\/jats:sup> 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\u2010ray crystal structure of the human enzyme showed the aldehyde oxygen atom 3.5\u2005\u00c5 away from the heme iron atom. Experiments with human P450 51A1 and H<jats:sub>2<\/jats:sub><jats:sup>18<\/jats:sup>O yielded primarily one <jats:sup>18<\/jats:sup>O atom but 14\u2009% of the formic acid product with two <jats:sup>18<\/jats:sup>O atoms, indicative of a minor contribution of a Compound I mechanism. LC\u2013MS evidence for a Compound 0\u2010derived Baeyer\u2013Villiger reaction product (a 14\u03b1\u2010formyl ester) was also found.<\/jats:p>","DOI":"10.1002\/anie.202317711","type":"journal-article","created":{"date-parts":[[2024,1,11]],"date-time":"2024-01-11T12:31:10Z","timestamp":1704976270000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":26,"title":["Oxygen\u201018 Labeling Reveals a Mixed Fe\u2212O Mechanism in the Last Step of Cytochrome P450 51 Sterol 14\u03b1\u2010Demethylation"],"prefix":"10.1002","volume":"63","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0027-9120","authenticated-orcid":false,"given":"Kevin D.","family":"McCarty","sequence":"first","affiliation":[{"name":"Department of Biochemistry Vanderbilt University School of Medicine  Nashville TN 37232\u20130146 USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1884-014X","authenticated-orcid":false,"given":"Yasuhiro","family":"Tateishi","sequence":"additional","affiliation":[{"name":"Department of Biochemistry Vanderbilt University School of Medicine  Nashville TN 37232\u20130146 USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tatiana Y.","family":"Hargrove","sequence":"additional","affiliation":[{"name":"Department of Biochemistry Vanderbilt University School of Medicine  Nashville TN 37232\u20130146 USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6975-1131","authenticated-orcid":false,"given":"Galina I.","family":"Lepesheva","sequence":"additional","affiliation":[{"name":"Department of Biochemistry Vanderbilt University School of Medicine  Nashville TN 37232\u20130146 USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7458-3048","authenticated-orcid":false,"given":"F. 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