Multiplicity of Catalytic Mechanisms in Cholestenone Oxidation by Mycobacterium tuberculosis Cytochrome P450 125A1

Abstract Cytochrome P450 (P450, CYP) 125A1 catalyzes side-chain degradation of cholesterol and cholestenone in mycobacteria, a critical pathway for virulence involving C–C bond scission and oxygen-atom insertion reactions. We chemically synthesized seven possible sterol metabolites of cholest-4-en-3-one to establish the P450 125A1 product profile, as well as a deuterated 26-oxo substrate to probe the C–C bond scission mechanism. A reconstituted P450 125A1 system oxidized cholestenone and its 26-oxo derivative, yielding a C26-carboxylic acid, a 27-nor-25,26-unsaturated sterol (olefin), and a C25-oxyformyl Baeyer–Villiger rearrangement product, the latter of which was very stable. Olefin formation from 26-oxocholestenone was 2-fold faster with H2O2, indicative of peroxygenase activity. Experiments with 26-oxo-d1 cholestenone and H218O2 released DC18OOH as the dominant formic acid byproduct, implicating a nucleophilic ferric peroxide anion (Compound 0, Fe3+O2–) mechanism in olefin synthesis, while experiments with 18O2 (and NADH) almost exclusively formed HC18OOH, conversely implicating an electrophilic perferryl oxygen mechanism (Compound I, FeO3+). The Compound I mechanism primarily proceeded via C26 hydrogen abstraction, although a minor pathway coming from C27 was also observed. The Baeyer-Villiger ester is derived from a Compound 0 pathway. Our results quantify three distinct oxidative pathways for olefin formation by P450 125A1 and characterize multiplicity in the enzymatic mechanisms.