Cross-linking of human cytochrome P450 2B6 to NADPH-cytochrome P450 reductase: Identification of a potential site of interaction

J Inorg Biochem. 2010 Apr;104(4):485-8. doi: 10.1016/j.jinorgbio.2009.12.017. Epub 2010 Jan 4.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Binding Sites
  • Carbodiimides / chemistry
  • Cross-Linking Reagents / chemistry
  • Cytochrome P-450 CYP2B6
  • Cytochrome P-450 Enzyme System / chemistry*
  • Cytochrome P-450 Enzyme System / genetics
  • Humans
  • Mass Spectrometry
  • Molecular Sequence Data
  • NADPH-Ferrihemoprotein Reductase / chemistry*
  • NADPH-Ferrihemoprotein Reductase / genetics
  • Peptides / chemistry
  • Peptides / genetics
  • Protein Binding


  • Carbodiimides
  • Cross-Linking Reagents
  • Peptides
  • Cytochrome P-450 Enzyme System
  • CYP2B6 protein, human
  • Cytochrome P-450 CYP2B6
  • NADPH-Ferrihemoprotein Reductase