The cytochromes P450 are a super family of isozymes that participate in a wide range of essential metabolic reactions, including the synthesis of steroids and the degradation of exogenous compounds such as polyaromatic hydrocarbons. The P45Os, in combination with NADPH cytochrome P450 reductase (reductase) and cytochrome b5 (b5), are part of a multiprotein system in the endoplasmic reticulum that requires the transfer of electrons from reductase or b5 to the P450. Extensive studies have been done to understand the various P450 families that are involved in the metabolism of xenobiotics, since there is strong evidence that their enzymatic actions on certain substrates can lead to the formation of promutagens and procarcinogens. There is conflicting evidence concerning the nature of the protein-protein interactions that regulate this system. We intend to resolve these issues by conducting a systematic investigation of the protein assembly reactions of P450, reductase, and b5. We will focus on the 2E1 isozyme of the P450 super family. P450 2E1 is responsible for the metabolism of ethanol and other small exogenous compounds and has been implicated in many alcohol-related diseases as well as carcinogenesis. We will study the complexes formed by the proteins purified from rat liver. P450 2B1 will also be investigated to assess potential differences between P450 isozymes. Besides examining the role(s) of protein-protein interactions, we will also probe the role(s) of protein-lipid interactions and the membrane. The composition of all homo- and heterocomplexes that can form from these three proteins will be determined using fluorescence techniques and analytical ultracentrifugation. The hydrodynamic properties of the complexes will be evaluated to obtain a low resolution picture of their global shapes to help establish how they associate. These studies will involve our recently demonstrated approach of refining the solution structure and dynamics of a protein complex by combining time-resolved fluorescence anisotropy with sedimentation velocity. We will also determine the specific roles that electrostatic, van der Waals, and hydrophobic interactions have in the complexes. The nature of the interactions will be examined by varying thermodynamic parameters and measuring changes in the dissociation constant, size, and shape of each complex. The orientation of a particular protein in a complex will be evaluated by fluorescence quenching and anisotropy changes of probes at unique sites. Thermodynamic and orientation information will then be correlated with the structures known for P45O(cam), P450BM-3, and bovine liver b5 to help define what portions of each protein interact with each other. Our comprehensive approach to this problem will clarify the nature of the biologically important interactions that P450 2E1 encounters. It will also be the basis for future studies on the other proteins in the electron transport system.
