Single-Protein Tracking Reveals That NADPH Mediates the Insertion of Cytochrome P450 Reductase into a Biomimetic of the Endoplasmic Reticulum.

Cytochrome P450 reductase (CPR) is the redox partner for most human cytochrome P450 enzymes. It is also believed that CPR is an integral membrane protein exclusively. Herein, we report that, contrary to this belief, CPR can exist as a peripheral membrane protein in the absence of NADPH and will transition to an integral membrane protein in the presence of stoichiometric amounts of NADPH or greater. All experiments were performed in a solid-supported cushioned lipid bilayer that closely matched the chemical composition of the human endoplasmic reticulum and served as an ER biomimetic. The phase characteristics and fluidity of the ER biomimetic was characterized with fluorescence micrographs and temperature-dependent fluorescence recovery after photobleaching. The interactions of CPR with the ER biomimetic were directly observed by tracking single CPR molecules using time-lapse single-molecule fluorescence imaging and subsequent analysis of tracks. These studies revealed dramatic changes in diffusion coefficient and the degree of partitioning of CPR as a function of NADPH concentration.