The first step in the biosynthesis of steroid hormones such as cortisol, testosterone, progesterone, and estrogen is catalyzed by the enzyme cytochrome P450 11A1 (P450 11A1). Starting with cholesterol, this enzyme first adds two hydroxyl groups (each containing an oxygen and a hydrogen atom) to two adjacent carbons of the molecule. Then it cleaves the bond between those two atoms. The product of this reaction is called pregnenolone. The exact mechanism by which P450 11A1 uses oxygen from the atmosphere to add the two hydroxyl groups to cholesterol has been thoroughly studied and is now reasonably well understood. However, considerable controversy remains regarding exactly how the enzyme cleaves the carbon-to-carbon bond. Two distinct hypotheses are consistent with what is currently known about P450 enzymes, leading Vanderbilt Basic Sciences investigator Fred Guengerich and his laboratory to launch a series of elegant experiments to determine which hypothesis is correct. Using a combination of chemical synthesis, isotopic labeling, and high-resolution mass spectrometry, the investigators were able to show that the reaction starts when the one of the hydroxyl groups that had been added to the cholesterol substrate attacks the iron (IV) oxo group of the heme in the enzyme’s active site. This work sheds new light into the mechanism of action of an important hormone biosynthetic enzyme and is also applicable to other P450 enzymes that are responsible for metabolizing 90% of all drugs. The work is published in the Journal of the American Chemical Society [F. K. Yoshimoto, J. Am. Chem. Soc., 138, 12124].