Ph.D., Senior Research Biologist, Department of Molecular and Integrative Physiology, UIUC

Molecular organization of microsomal drug metabolizing enzymes, cytochrome P450s, and formation of heteromeric complexes with P450 reductase have been mostly studied with the isolated proteins and using reconstituted systems.  These studies suggested the existence of some P450s in multimeric complexes, however, it is not known whether these proteins form oligomers in cells,  whether self-association characterizes all P450s and what complexes do they form with the P450 reductase.  We have used fluorescence resonance energy transfer (FRET) technique to study P450 oligomerization and reductase binding in live transfected cells.  In addition to the emission scanning spectrofluorimetry, we have applied two methods to detect FRET in live cells using confocal microscopy: ratio imaging and acceptor photobleaching.   The results indicate that cytochrome P450 2C2, which is statically retained in the membranes of the endoplasmic reticulum (ER), forms homooligomeric structures and this self-association is mediated by the transmembrane sequence.  In contrast, cytochrome P450 2E1, which can escape ER retention, does not form homooligomeric structures, nor does it associate with P450 2C2.  Using FRET, we have been able to detect reductase binding by both, oligomers forming P450 2C2 and monomeric P450 2E1. These results suggest that microsomal P450s may differ in their quarternary structure, however, there is no direct correlation between oligomerization and static retention in the endoplasmic reticulum.