Cytochrome P450 aromatase is a membrane bound enzyme that synthesises estrogens. 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. 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. Forster resonance energy transfer (FRET) studies explored the protein-protein interactions in the ER. A quartz crystal microbalance (QCM) 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. Molecular mechanics calculations were also used to further probe the human and porcine P450arom.
Our FRET studies showed that the human P450arom forms dimers in vivo. The QCM showed that all the recombinant P450arom enzymes examined bound tightly to the lipid membranes; both truncated and full length. The P450arom: CPR complexes bound to the membranes exhibited good catalytic turnover. However, the human P450arom associated very differently with the lipid membrane than the porcine gonadal P450arom. The rate of porcine P450arom binding was most influenced by the amount of CPR present. Thus the structural organisation within the membrane is very different between porcine and human, despite the similarities in amino acid sequence. The minimum energy structures for the human and porcine gonadal P450arom also differ and thus may influence the mechanism of P450arom function.
Declaration of interest: The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project.
Funding: This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.