Isotope-Labeling Studies Support the Electrophilic Compound I Iron Active Species, FeO3+, for the Carbon–Carbon Bond Cleavage Reaction of the Cholesterol Side-Chain Cleavage Enzyme, Cytochrome P450 11A1

The enzyme cytochrome P450 11A1 cleaves the C20–C22 carbon–carbon bond of cholesterol to form pregnenolone, the first 21-carbon precursor of all steroid hormones. Various reaction mechanisms are possible for the carbon–carbon bond cleavage step of P450 11A1, and most current proposals involve the oxoferryl active species, Compound I (FeO3+). Compound I can either (i) abstract an O–H hydrogen atom or (ii) be attacked by a nucleophilic hydroxy group of its substrate, 20R,22R-dihydroxycholesterol. The mechanism of this carbon–carbon bond cleavage step was tested using 18O-labeled molecular oxygen and purified P450 11A1. P450 11A1 was incubated with 20R,22R-dihydroxycholesterol in the presence of molecular oxygen (18O2), and coupled assays were used to trap the labile 18O atoms in the enzymatic products (i.e., isocaproaldehyde and pregnenolone). The resulting products were derivatized and the 18O content was analyzed by high-resolution mass spectrometry. P450 11A1 showed no incorporation of an 18O atom into e...