Modulation of the cytochrome P450 reductase redox potential by the phospholipid bilayer

Abstract

Cytochrome P450 reductase (CPR) is a tethered membrane protein which transfers electrons from NADPH to microsomal P450s. We show that the lipid bilayer has a role in defining the redox potential of the CPR flavin domains. In order to quantitate the electrochemical behavior of this central redox protein, full-length CPR was incorporated into soluble nanometer scale discoidal membrane bilayers (nanodiscs), and potentials were measured using spectropotentiometry. The redox potentials of both FMN and FAD were found to shift to more positive values when in a membrane bilayer as compared to a solubilized version of the reductase. The potentials of the semiquinone/hydroquinone couple of both FMN and FAD are altered to a larger extent than the oxidized/semiquinone couple which is understood by a simple electrostatic model. When anionic lipids were used to change the membrane composition of the CPR-nanodisc, the redox potential of both flavins became more negative, favoring electron transfer from CPR to cytochrome P450.

Scheme 1

Structure of flavin redox states.

Figure 1

(A) Schematic representation of CPR docking in the lipid bilayer membrane of Nanodiscs (B) The HPLC elution profiles of CPR-Nanodisc assemblies following the flavin absorbances at 453 nm. A Stokes diameter scale is shown on the top axis.

Figure 2

Spectral changes during a redox titration of full-length CPR in Nanodiscs.

Figure 3

Multiple wavelength analysis of the redox titration of CPR-Nanodiscs assembled with POPC. The absorbance is plotted versus the solution potential (mV vs. SHE) at several wavelength regions: (A) 450-460 nm, (B) the isosbestic of semiquinone/reduced couple at 429 nm, (C) 580-605 and (D) the isosbestic of the oxidized/semiquinone at 501 nm. The solid lines represent fits to the four electron Nernst equation (Methods).

Figure 4

Multiple wavelength analysis of the redox titration of CPR-Nanodiscs with 50% POPS and 50% POPC. (A-D) correspond to the wavelengths of absorption measurements as described in Figure 3.

Figure 5

Effect of anionic lipids on the CPR redox potentials. Potential CPR-Nanodiscs with 100% POPC (open circles) is shifted to higher values as compared to CPRNanodiscs containing 50% POPC/50% POPS (open squares).