Cytochrome P450aromatase is a membrane bound enzyme in vertebrates that catalyses the synthesis of estrogens from androgens. This reaction involves electron delivery from NADPH via cytochrome P450 reductase (CPR).  Little is known as to how the P450arom performs the aromatase reaction or how it associates with CPR in the endoplasmic reticulum (ER). In humans, there is only one P450arom, however pigs have three isozymes.¹ The catalytic efficiency of one of these, the porcine gonadal P450arom, is much lower than the human and the porcine placental isozymes despite the high amino acid sequence homologies.

We have used in vivo and in vitro techniques to study the interaction of P450arom proteins and lipid membranes. FRET (Forster Resonance Energy Transfer) studies explored the protein-protein interactions in the ER. A QCM (quartz crystal microbalance) was used to measure the mass of protein(s) binding to a lipid membrane and in association with the Western blot data the stoichiometry for P450arom and CPR was determined. QCM also provides information about the conformational and structural organisation of proteins in the lipid membrane. In silico calculations were also used to further probe the human placental and porcine gonadal P450arom.

Our FRET studies showed that the human P450arom forms dimers in vivo.² The QCM showed tight binding of all recombinant P450arom enzymes examined to the lipid membranes.²  The reconstituted P450arom:CPR complexes exhibited high catalytic rates. However, the human P450arom ‘associated’ very differently with the lipid membrane than the porcine gonadal P450arom did.  The rate of porcine P450arom binding was most influenced by the amount of CPR present. Thus, despite their high homology the structural organisation of each P450arom within the membrane differs. The solvation energy of the respective human and porcine gonadal P450arom dimer also differs, indicating that dimerization may influence the mechanism of P450arom function.