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 18O–water based on the principle that the cross-linked peptides would be expected to incorporate twice as many 18O 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.

Graphical abstractIsotopic labeling of cross-linked peptides coupled with mass spectrometry was used to identify P450 2B6 residues that potentially interact with NADPH-cytochrome P450 reductase. The identified amino acids lie in what is predicted to be the C/D loop of P450 2B6 based on the X-ray crystal structure of P450 2B4.Figure optionsDownload full-size imageDownload as PowerPoint slide