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CYP19A1, or aromatase, a cytochrome P450 responsible for estrogen biosynthesis in humans, is an important therapeutic target for the treatment of breast cancer. There is still controversy surrounding the identity of reaction intermediate that catalyzes carbon–carbon scission in this key enzyme. Probing the oxy-complexes of CYP19A1 poised for hydroxylase and lyase chemistries using resonance Raman spectroscopy and drawing a comparison with CYP17A1, we have found no significant difference in the frequencies or isotopic shifts for these two steps in CYP19A1. Our experiments implicate the involvement of Compound I in the terminal lyase step of CYP19A1 catalysis.

SUMMARY Cytochromes P450 from Eukaryotes and their native redox partners cytochrome P450 reductases both belong to the class of monotopic membrane proteins containing one transmembrane anchor. Incorporation into the lipid bilayer significantly affects their equilibrium and kinetic properties and plays an important role in their interactions. We describe here the detailed protocols developed in our group for the functional self-assembly of mammalian cytochromes P450 and cytochrome P450 reductases into Nanodiscs with controlled lipid composition. The resulting preparations are fully functional, homogeneous in size, composition and oligomerization state of the heme enzyme, and show an improved stability with respect to P420 formation. We provide a brief overview of applications of Nanodisc technology to the biophysical and biochemical mechanistic studies of cytochromes P450 involved in steroidogenesis, and of the most abundant xenobiotic metabolizing human cytochrome P450 CYP3A4.

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.