期刊
JOURNAL OF INORGANIC BIOCHEMISTRY
卷 104, 期 4, 页码 485-488出版社
ELSEVIER SCIENCE INC
DOI: 10.1016/j.jinorgbio.2009.12.017
关键词
Cytochrome P450; Cross-linking; Protein-protein interactions
资金
- National Cancer Institute, National Institutes of Health [CA16954]
- National Institutes of Health [T32-GM007767]
- PhRMA Foundation
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 O-18-water based on the principle that the cross-linked peptides would be expected to incorporate twice as many O-18 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. (C) 2009 Elsevier Inc. All rights reserved.
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