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Structural and Functional Studies of the Membrane-Binding Domain of NADPH-Cytochrome P450 Oxidoreductase
journal contribution
posted on 2019-04-22, 00:00 authored by Chuanwu Xia, Anna L. Shen, Panida Duangkaew, Rattanawadee Kotewong, Pornpimol Rongnoparut, Jimmy Feix, Jung-Ja P. KimNADPH-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
cytochrome 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. We show that the MBD spans the membrane
and present a model for the 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. The
deletion of more than 29 residues from the N-terminus of CYPOR decreases
cytochrome P450 activity concomitant with alterations in electrophoretic
mobility and an increased resistance to protease digestion. The altered
kinetic properties of these mutants are consistent with electron transfer
through random collisions rather than via formation of a stable CYPOR-P450
complex. Purified MBD binds weakly to cytochrome P450, suggesting
that other interactions are also required for CYPOR-P450 complex formation.
We propose that the MBD and flexible tether region of CYPOR, residues
51–63, play an important role in facilitating the movement
of the soluble domain relative to the membrane and in promoting multiple
orientations that permit specific interactions of CYPOR with its varied
partners.
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Keywords
amino-terminal deletion mutantsP 450 catalysisNADPH-Cytochrome P 450 Oxidoreductase NADPH-cytochrome P 450 oxidoreductasecytochrome P 450 monooxygenase systemelectron transfercytochrome P 450. EPR power saturation measurementsamino-terminal membrane-binding domainMBDroleCYPOR decreases cytochrome P 450 activityCYPOR-P 450cytochrome P 450.
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