Kinetic solvent isotope effect in steady-state turnover by CYP19A1 suggests involvement of Compound 1 for both hydroxylation and aromatization steps

•The KSIE of the hydroxylation and lyase reactions by human CYP19A1 were investigated.•Compound 1 is involved in first two steps of hydroxylation.•The most controversial C–C lyase reaction (step III) also uses the Compound 1 intermediate. CYP19A1, or human aromatase catalyzes the conversion of androgens to estrogens in a three-step reaction through the formation of 19-hydroxy and 19-aldehyde intermediates. While the first two steps of hydroxylation are thought to proceed through a high-valent iron-oxo species, controversy exists surrounding the identity of the reaction intermediate that catalyzes the lyase and aromatization reaction. We investigated the kinetic isotope effect on the steady-state turnover of Nanodisc-incorporated human CYP19A1 to explore the mechanisms of this reaction. Our experiments reveal a significant (∼2.5) kinetic solvent isotope effect for the C10–C19 lyase reaction, similar to that of the first two hydroxylation steps (2.7 and 1.2). These data implicate the involvement of Compound 1 as a reactive intermediate in the final aromatization step of CYP19A1.