Fluorescence Resonance Energy Transfer Analysis of Cytochromes P450 2C2 and 2E1 Molecular Interactions in Living Cells

Abstract

The molecular organization of microsomal cytochromes P450 (P450s) and formation of complexes with P450 reductase have been studied previously with isolated proteins and in reconstituted systems. Although these studies demonstrated that some P450s oligomerize in vitro, neither oligomerization nor interactions of P450 with P450 reductase have been studied in living cells. Here we have used fluorescence resonance energy transfer (FRET) to study P450 oligomerization and binding to P450 reductase in live transfected cells. Cytochrome P450 2C2, but not P450 2E1, forms homo-oligomeric structures, and this self-association is mediated by the signal-anchor sequence. Because P450 2C2, in contrast to P450 2E1, is directly retained in the endoplasmic reticulum (ER), these results could suggest that oligomerization may prevent transport from the ER. However, P450 2C1 signal-anchor sequence chimera defective in ER retention also formed oligomers, and chimera containing the cytoplasmic domain of P450 2C2, which is directly retained in the ER, did not exhibit self-oligomerization, which indicates that oligomerization is not correlated with direct retention. By using FRET, we have also detected binding of P450 2C2 and P450 2E1 to P450 reductase. In contrast to self-oligomerization, the catalytic domain can mediate an interaction of P450 2C2 with P450 reductase. These results suggest that microsomal P450s may differ in their quaternary structure but that these differences do not detectably affect interaction with the reductase or transport from the ER.The organization of the cytochrome P450 (P450) 1 -containing monooxygenase system in the microsomal membrane is not well understood despite many studies using a wide variety of biophysical methods. This system consists of P450s, NADPHcytochrome P450 reductase (P450 reductase), and for some P450s, cytochrome b 5 . If P450 levels in the endoplasmic reticulum (ER) are induced to maximal concentrations, there are 20 -30 P450s for each P450 reductase molecule. The presence of multiple forms of P450s in the ER membranes combined with limiting amounts of P450 reductase has important implications for the association of the P450s with each other and the reductase and for electron transfer from the reductase to P450. It has been postulated that either a multimeric complex of P450s binds to a molecule of P450 reductase (1, 2) or that the interaction results from random collisions of independently diffusing monomeric proteins that have high rotational and lateral mobility (3-5). Formation of large complexes by P450s has been observed with both isolated proteins and reconstituted systems (6 -8). Studies on rotational mobilities of P450s in microsomal membranes were also consistent with either self-aggregation or association of P450s with other components of the membranes (9 -11). The second model in which monomeric P450 interacts with P450 reductase is supported by observations that high concentrations of some P450s in membranes result in their aggregation and immobilization, but the addition of...

Effect of glutathione on homo- and heterotropic cooperativity in cytochrome P450 3A4

“…A similar mechanism may also take place in the membrane, where microsomal cytochromes P450 are also known to exist in equilibrium between monomers and oligomers [67][68][69][70][71][72]. However, the reversibility of oligomerization in the membrane, as opposed to virtual irreversibility in solution, would blunt the manifestations of the proposed conformational heterogeneity in the membrane-bound enzyme.…”

Heteromeric Complex Formation between CYP2E1 and CYP1A2: Evidence for the Involvement of Electrostatic Interactions

“…CYP2E1 was shown to exist as a 10-mer in solution that could not be dissociated by high concentrations of detergent (42). Szczesna-Skorupa et al (43) showed that CYP2E1 does not self-associate as measured by fluorescence resonance energy transfer (FRET) in cell culture, although CYP2E1 was shown to complex with reductase. In contrast, homomeric complex formation was reported for CYP2C2 using this technique (43).…”

“…Szczesna-Skorupa et al (43) showed that CYP2E1 does not self-associate as measured by fluorescence resonance energy transfer (FRET) in cell culture, although CYP2E1 was shown to complex with reductase. In contrast, homomeric complex formation was reported for CYP2C2 using this technique (43). Again, complex formation between CYP2C2 and reductase was observed.…”

Role of subunit interactions in P450 oligomers in the loss of homotropic cooperativity in the cytochrome P450 3A4 mutant L211F/D214E/F304W

“…This functional heterogeneity appears to be linked closely to the oligomeric state of the enzyme [19,20]. Microsomal cytochromes P450 are known to form oligomers both in solution [25][26][27] and in membranes [28][29][30][31][32][33], and direct association between P450s has been recognized as an important determinant of function [34][35][36][37]. In particular, functional heterogeneity of a single P450 species caused by oligomerization has been revealed in interactions with substrates [19,38,39].…”