Quantum-Mechanical/Molecular-Mechanical Studies of CYP11A1-Catalyzed Biosynthesis of Pregnenolone from Cholesterol Reveal a C–C Bond Cleavage Reaction That Occurs by a Compound I-Mediated Electron Transfer

We explore here a long-standing mechanistic question by using quantum-mechanical/molecular-mechanical (QM/MM) methodology. The question concerns the mechanism of steroid hormone biosynthesis, whereby the P450 enzyme, CYP11A1, catalyzes the C20–C22 bond-cleavage in the 20,22-hydroxylated cholesterol, 20 R,22 R -DiOHCH, leading to pregnenolone, which is critical for the subsequent production of all steroid hormones . This is an unusual feat whereby the P450 enzyme breaks two O–H bonds and one C–C bond, while making two C═O bonds. How does the enzyme perform such a complex and highly energy-demanding reaction? Our computational results rule out the previously proposed Compound I (Cpd I) electrophilic attack mechanism via the formation of a peroxide intermediate as well as the H-abstraction-mediated C–C cleavage mechanism. Notably, oxygen-rebound cannot transpire, in spite of the fact that the classical active species, Cpd I, participates in the catalytic process. Our findings reveal a mechanism whereby C–C bond cleavage is mediated by an electron transfer from the C22–O – - deprotonated substrate to Cpd I . As such, our QM/MM calculations demonstrate that Cpd I acts as an electron sink that facilitates the C–C bond cleavage .