J._Biol._Chem.-2009-Ellis-36628-37[1].pdf (2.71 MB)
Download fileDomain motion in cytochrome P450 reductase: conformational equilibria revealed by NMR and small-angle X-Ray scattering
journal contribution
posted on 2010-06-04, 09:35 authored by Jacqueline Ellis, Aldo Gutierrez, Igor L. Barsukov, Wei-Cheng Huang, J. Günter Grossman, Gordon C. K. RobertsNADPH-cytochrome P450 reductase (CPR), a diflavin reductase,
plays a key role in the mammalian P450 mono-oxygenase
system. In its crystal structure, the two flavins are close together,
positioned for interflavin electron transfer but not for electron
transfer to cytochrome P450.Anumber of lines of evidence suggest
that domain motion is important in the action of the
enzyme. We report NMR and small-angle x-ray scattering experiments
addressing directly the question of domain organization
in human CPR. Comparison of the 1H-15N heteronuclear
single quantum correlation spectrum of CPR with that of the
isolatedFMNdomain permitted identification of residues in the
FMN domain whose environment differs in the two situations.
These include several residues that are solvent-exposed in the
CPR crystal structure, indicating the existence of a second conformation
in which the FMN domain is involved in a different
interdomain interface. Small-angle x-ray scattering experiments
showed that oxidized and NADPH-reduced CPRs have
different overall shapes. The scattering curve of the reduced
enzyme can be adequately explained by the crystal structure,
whereas analysis of the data for the oxidized enzyme indicates
that it exists as a mixture of approximately equal amounts of two
conformations, one consistent with the crystal structure and
one a more extended structure consistent with that inferred
from the NMR data. The correlation between the effects of
adenosine 2’,5’-bisphosphate and NADPH on the scattering
curve and their effects on the rate of interflavin electron transfer
suggests that this conformational equilibrium is physiologically
relevant.