Investigating the Effects and Mechanisms of Interactions between Cytochrome P450 2B4, Cytochrome P450 2E1 and Cytochrome P450 Reductase

A requirement for cytochrome P450 (CYP or P450)-mediated drug metabolism is the association of P450s with cytochrome P450 reductase (CPR). Although P450s form a 1:1 complex with CPR, they exist in excess over CPR in the endoplasmic reticulum (ER). Very little is known about the effect of less than stoichiometric amounts of CPR relative to P450 in the ER on the interaction of P450s with CPR and substrate metabolism. Equally little is known about the mechanism of interaction of P450s with CPR since much of our knowledge regarding the specific residues that mediate this interaction stems from a limited number of mutagenesis studies. In this thesis we developed methodology to directly probe the CYP2B4-CPR binding interface and demonstrated novel roles for residues V267 and L270 of CYP2B4 in binding CPR. We harnessed this knowledge to engineer a CYP2B4 with greater rates of reduction and substrate metabolism. We also found that CYP2E1, an inducible P450 isoform, significantly inhibited the catalytic activity of CYP2B4 in a concentration-dependent manner. We proposed a preliminary model to explain the inhibitory behavior of CYP2E1 toward CYP2B4 that was based on two key findings: 1) direct CYP2B4-CYP2E1 interactions alone do not lead to inhibition of CYP2B4 activity in the presence of saturating concentrations of substrate and 2) CYP2E1 has a higher affinity for CPR in the presence of CYP2B4. In this model we suggested that CYP2E1 and CYP2B4 associate to form a CYP2B4-CYP2E1 complex that interacts with the functional site of CPR with a higher affinity than CYP2E1 alone, and this complex may allow CYP2E1 to compete with CYP2B4 for CPR. Taken together, the work presented in this thesis establishes a new approach to the identification of amino acid residues that mediate redox-partner recognition and demonstrates how these residues can be used to enhance P450 activity. Additionally, these reports provide us with valuable insights into the potential for protein-protein interactions in the P450 system to confound in vitro – in vivo drug metabolism extrapolations and may play an important role in improving our ability to predict in vivo drug clearance and drug-drug interactions from in vitro data.PHDChemical BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/94027/1/ckenaan_1.pd

Reaction of Human Cytochrome P450 3A4 with Peroxynitrite: Nitrotyrosine Formation on the Proximal Side Impairs Its Interaction with NADPH-Cytochrome P450 Reductase

The reaction of peroxynitrite (PN) with purified human cytochrome P450 3A4 (CYP3A4) resulted in the loss of the reduced-CO difference spectrum, but the absolute absorption spectrum of the heme was not significantly altered. The loss of 7-benzyloxy-4-(trifluoromethyl)­coumarin (BFC) O-debenzylation activity of CYP3A4 was concentration-dependent with respect to PN, and the loss of BFC activity supported by NADPH-cytochrome P450 reductase (CPR) was much greater than that supported by <i>tert</i>-butyl hydroperoxide. Moreover, the PN-treated CYP3A4 exhibited a reduced-CO spectrum when reduced by CPR that was much smaller than when it was reduced by dithionite. These results suggest that modification of CYP3A4 by PN may impair its interaction with CPR, leading to the loss of catalytic activity. Tyrosine nitration, as measured by an increase in mass of 45 Da due to the addition of a nitro group, was used as a biomarker for protein modification by PN. PN-treated CYP3A4 was digested by trypsin and endoproteinase Glu C, and nitrotyrosine formation was then determined by using electrospray ionization–liquid chromatography–tandem mass spectrometry. Tyr residues 99, 307, 347, 430, and 432 were found to be nitrated. Using the GRAMM-X docking program, the structure for the CYP3A4–CPR complex shows that Tyr99, Tyr347, and Tyr430 are on the proximal side of CYP3A4 and are in close contact with three acidic residues in the FMN domain of CPR, suggesting that modification of one or more of these tyrosine residues by PN may influence CPR binding or the transfer of electrons to CYP3A4. Mutagenesis of Tyr430 to Phe or Val revealed that both the aromatic and the hydroxyl groups of Tyr are required for CPR-dependent catalytic activity and thus support the idea that the proximal side Tyr participates in the 3A4–CPR interaction. In conclusion, modification of tyrosine residues by PN and their subsequent identification can be used to enhance our knowledge of the structure/function relationships of the P450s with respect to the electron transfer steps, which are critical for P450 activity