Structural and Functional Studies of the Membrane Binding Domain of NADPH -Cytochrome P450 Oxidoreductase

NADPH-cytochrome P450 oxidoreductase (CYPOR), the essential flavoprotein of the microsomal cytochrome P450 monooxygenase system, is anchored in the phospholipid bilayer by its amino-terminal membrane-binding domain (MBD), which is necessary for efficient electron transfer to cytochromes P450. Although crystallographic and kinetic studies have established the structure of the soluble catalytic domain and the role of conformational motions in the control of electron transfer, the role of the MBD is largely unknown. We examined the role of the MBD in P450 catalysis through studies of amino-terminal deletion mutants and site-directed spin-labeling EPR. We show that the MBD spans the membrane and present a model for orientation of CYPOR on the membrane capable of forming a complex with cytochrome P450. EPR power saturation measurements of CYPOR mutants in liposomes containing a lipid-Ni(II)chelate identified a region of the soluble domain interacting with the membrane. Deletion of more than 29 residues from th...