Redox potential control by drug binding to cytochrome p450 3A4

The cytochrome P450s are ubiquitous heme proteins that utilize two reducing equivalents to cleave a ferrous iron-dioxygen complex to produce a single water molecule with the Insertion of one oxygen atom into a bound substrate. For the case of soluble cytochrome P450 CYP101, it has been shown that there is a linear free-energy relationship between heme redox potential and the spin state of the ferric protein. However, the universality of this relationship has been challenged in the case of mammalian enzymes. Most cytochrome P450s are integral membrane proteins, and detailed redox potential measurements have proved difficult because of protein aggregation or the necessary presence of detergent. In this Communication we utilize a soluble nanometer scale membrane bilayer disc (Nanodisc) to stabilize monomeric human cytochrome P450 CYP3A4. The Nanodisc system allows facile redox potential measurements to be made on substrate-free CYP3A4 as well as with several drug molecules bound at the active site. We show that substrate binding can dramatically effect the redox potential of the heme protein through modulation of the ferric spin state. A linear free-energy relationship is observed, analogous to that noted for the soluble P450s, indicating a common mechanism for this linkage and providing a means for control of electron input in response to the presence of a metabolizable substrate, this potentially limiting the unwanted production of reduced oxygen species.