Cross-linking of human cytochrome P450 2B6 to NADPH-cytochrome P450 reductase: Identification of a potential site of interaction.
The site(s) of interaction between human cytochrome P450 2B6 and NADPH-cytochrome P450 reductase (P450 reductase) have yet to be identified. To investigate this, the cross-linking agent 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) was used to covalently link P450 2B6-P450 reductase. Following digestion with trypsin, the cross-linked peptides were identified by reconstituting the peptides in (18)O-water based on the principle that the cross-linked peptides would be expected to incorporate twice as many (18)O atoms as the non-cross-linked peptides. Subsequent mass spectrometric analyses of the resulting peptides led to the identification of one cross-linked peptide candidate. De novo sequencing of the peptide indicated that it is a complex between residues in the C-helix of the P450 (based upon solved X-ray crystal structures of P450 2B4) and the connecting domain of the P450 reductase. To confirm this experimentally, the P450 2B6 peptide identified through the cross-linking studies was synthesized and peptide competition studies were performed. In the presence of the synthetic peptide, P450 catalytic activity was decreased by up to 60% when compared to competition studies performed using a nonsense peptide. Taken together, these studies indicate that residues in the C-helix of P450 2B6 play a major role in the interaction with the P450 reductase.
Bumpus
Namandjé N
NN
Department of Pharmacology, University of Michigan, Ann Arbor, MI 48109-5632, United States.
Hollenberg
Paul F
PF
D000595:Amino Acid Sequence; D001665:Binding Sites; D002234:Carbodiimides / Q000737:chemistry; D003432:Cross-Linking Reagents / Q000737:chemistry; D065702:Cytochrome P-450 CYP2B6; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry* / Q000235:genetics; D010455:Peptides / Q000737:chemistry / Q000235:genetics; D011485:Protein Binding
D002234:Carbodiimides; D003432:Cross-Linking Reagents; D010455:Peptides; D003577:Cytochrome P-450 Enzyme System; C585599:CYP2B6 protein, human; D065702:Cytochrome P-450 CYP2B6; D009251:NADPH-Ferrihemoprotein Reductase