Formation of raloxifene homo-dimer in CYP3A4, evidence for multi-substrate binding in a single catalytically competent P450 active site

Arch Biochem Biophys. 2011 Sep 15;513(2):110-8. doi: 10.1016/ Epub 2011 Jul 13.


Raloxifene is a polyaromatic compound which has been reported to form radicals when incubated with horseradish peroxidase resulting in formation of a homo-dimer product. Polyaromatic phenols have also been reported to undergo oxidation by P450 enzymes to form reactive intermediates, presumably through the formation of phenoxy radical species. Recently, we observed that a raloxifene homo-dimer was formed in vitro when incubated with CYP3A4. In response to this finding, a series of experiments were designed to determine whether the observed raloxifene homo-dimer was formed via solution phase chemistry similar to that previously documented with horseradish peroxidase or if generation of the homo-dimer occurred within the P450 active site. To this end, a series of experiments were carried out to determine the structure of the CYP3A4 generated raloxifene homo-dimer using analytical techniques including: high resolution MS, NMR and H/D exchange. In addition, a variety of in vitro techniques were applied to characterize the mechanism responsible for formation of the raloxifene homo-dimer. Collectively, the results of these experiments suggest that unlike the homo-dimer formed by peroxidase enzymes, raloxifene homo-dimer formation mediated by CYP3A4 is a consequence of two raloxifene molecules binding simultaneously within the active site of a catalytically competent P450 enzyme.

MeSH terms

  • Catalytic Domain
  • Cytochrome P-450 CYP3A / chemistry*
  • Cytochrome P-450 CYP3A / metabolism*
  • Deuterium Exchange Measurement
  • Dimerization
  • Electrochemical Techniques
  • Horseradish Peroxidase / chemistry
  • Horseradish Peroxidase / metabolism
  • Humans
  • Mass Spectrometry
  • Models, Molecular
  • Nuclear Magnetic Resonance, Biomolecular
  • Raloxifene Hydrochloride / chemistry*
  • Raloxifene Hydrochloride / metabolism*


  • Raloxifene Hydrochloride
  • Horseradish Peroxidase
  • Cytochrome P-450 CYP3A
  • CYP3A4 protein, human