Investigation of Surface Interactions between Cytochrome b5 and Major Cytochrome P450 Isoforms

Thesis (Ph.D.)--University of Washington, 2012Cytochrome b5 (cyt b5 or holo b5) is known as one of the key components in the microsomal cytochrome P450 (CYP) monooxygenase system that metabolizes structurally diverse endogenous and exogenous compounds. It has been reported to modulate many CYPs activity and the effect is both CYP isoform and substrate dependent. However, to date no consensus has been made on the underlying mechanism. In the present study, the surface interactions between cyt b5 and major hepatic CYP isoforms 3A4, 2C9, 2A6 and 2D6 were investigated. Chemical cross-linking coupled with mass spectrometric analysis was used to identify the potential electrostatic interactions on protein surfaces of cyt b5 and CYPs. Subsequently, the interaction models of cyt b5-CYPs were built using these identified cross-linking sites as constraints. For the first time, holo b5 and apo b5 (cyt b5 devoid of heme) surface interactions with CYPs were compared. The models suggest both of them bind to the same the groove on CYPs with very small difference in their orientations. The closest distances between the heme groups of cyt b5 and each CYP isoform are beyond direct electron transfer distance, indicating that cyt b5 likely modulates these CYP isoforms activity through allosteric effect in addition to the potential electron transfer role. In order to confirm that the residues involved in cross-linking are functionally important for cyt b5-CYP interaction, site-directed mutagenesis of CYP3A4 were carried out with the identified Lys residues on CYP3A4 being substituted with neutral residue Ala. In addition, the importance of Arg446 on CYP3A4 at the interface of the cyt b5-CYP3A4 complex model was also accessed by single-point mutation. Mutation of these residues reduced or abolished cyt b5 binding affinity, suggesting that electrostatic interactions on the interface of the two protein are functionally important and the chemical cross-linking coupled with mass spectrometric analysis serves as a useful tool to study protein-protein interaction