Characterization on the Structure and Dynamics of Protein-Protein Complexes Between Cytochrome P450 and its Redox Partners.

[摘要] Cytochrome P450s are a ubiquitous superfamily of monooxygenases responsible for the metabolism of thousands of compounds, including steroids, hormones, vitamins, and over 70% of the pharmaceuticals on the current market. They have been found to be related to human diseases including breast cancer and brain cancer. Therefore, it is of great importance to better understand the mechanism of how these enzymes function. In order for cytochrome P450s to complete the catalytic cycle, two electrons are required to be sequentially delivered to cytochrome P450 from its redox partners – cytochrome P450 reductase and cytochrome b5. Mammalian cytochrome P450s and redox partners are membrane bound proteins mostly found on the cytoplasmic side of the endoplasmic reticulum membrane. In order to provide insights into these essential electron-transfer steps, it is important to better understand complex formation between cytochrome P450 and its redox partners. In this thesis, complex formation between cytochrome P450 and cytochrome b5 is investigated under the effect of substrates and membrane. It is found that substrates and lipid bilayer facilitates stronger and more specific binding between the two proteins. Better membrane mimetics are developed in order to provide more physiologically relevant environment for future study on the cytochrome P450 system. A protein-protein complex structure is simulated using a docking program HADDOCK for the FMN binding domain of cytochrome P450 reductase and cytochrome P450. An electron transfer pathway is predicted based on this complex structure. This study provides better understanding on the interaction, binding interface and electron transfer process between the FMN binding domain of cytochrome P450 reductase and cytochrome P450. Substrates are found not to perturb the interaction between these two proteins, but affect the interplay of the three proteins when all of them are present in the same system, unravelling a competitive binding mechanism between cytochrome P450 reductase and cytochrome b5 for binding to cytochrome P450. This result provides structural explanation on the perplexing roles that cytochrome b5 plays in the cytochrome P450 activity and better understanding of substrate modulation on the tertiary protein complex system.

[发布机构] University of Michigan

[效力级别] cytochrome P450

[关键词] membrane protein;cytochrome P450;cytochrome P450 reductase;cytochrome b5;membrane;NMR;Biological Chemistry;Science;Chemistry