title: Insights into the role of substrates on the interaction between cytochrome b5 and cytochrome P450 2B4 by NMR.
abstract: Mammalian cytochrome b5 (cyt b5) is a membrane-bound protein capable of donating an electron to cytochrome P450 (P450) in the P450 catalytic cycle. The interaction between cyt b5 and P450 has been reported to be affected by the substrates of P450; however, the mechanism of substrate modulation on the cyt b5-P450 complex formation is still unknown. In this study, the complexes between full-length rabbit cyt b5 and full-length substrate-free/substrate-bound cytochrome P450 2B4 (CYP2B4) are investigated using NMR techniques. Our findings reveal that the population of complexes is ionic strength dependent, implying the importance of electrostatic interactions in the complex formation process. The observation that the cyt b5-substrate-bound CYP2B4 complex shows a weaker dependence on ionic strength than the cyt b5-substrate-free CYP2B4 complex suggests the presence of a larger fraction of steoreospecific complexes when CYP2B4 is substrate-bound. These results suggest that a CYP2B4 substrate likely promotes specific interactions between cyt b5 and CYP2B4. Residues D65, V66, T70, D71 and A72 are found to be involved in specific interactions between the two proteins due to their weak response to ionic strength change. These findings provide insights into the mechanism underlying substrate modulation on the cyt b5-P450 complexation process.
authors: Department of Chemistry and Biophysics, University of Michigan, Ann Arbor, Michigan 48109-1055, USA.
authors: Meng
authors: M
authors: Zhang
authors: Department of Chemistry and Biophysics, University of Michigan, Ann Arbor, Michigan 48109-1055, USA.
authors: Stéphanie V
authors: SV
authors: Le Clair
authors: Department of Chemistry and Biophysics, University of Michigan, Ann Arbor, Michigan 48109-1055, USA.
authors: Rui
authors: R
authors: Huang
authors: Department of Chemistry and Biophysics, University of Michigan, Ann Arbor, Michigan 48109-1055, USA.
authors: Shivani
authors: S
authors: Ahuja
authors: Department of Anesthesiology, University of Michigan, and VA Medical Center, Ann Arbor, Michigan 48105, USA.
authors: Sang-Choul
authors: SC
authors: Im
authors: Department of Anesthesiology, University of Michigan, and VA Medical Center, Ann Arbor, Michigan 48105, USA.
authors: Lucy
authors: L
authors: Waskell
authors: Department of Chemistry and Biophysics, University of Michigan, Ann Arbor, Michigan 48109-1055, USA.
authors: Ayyalusamy
authors: A
authors: Ramamoorthy
mesh_terms: D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry* / Q000378:metabolism; D000072467:Cytochrome P450 Family 2; D015786:Cytochromes b5 / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D009994:Osmolar Concentration; D011485:Protein Binding; D011487:Protein Conformation / Q000187:drug effects; D012965:Sodium Chloride / Q000494:pharmacology; D013379:Substrate Specificity
chemical_list: D012965:Sodium Chloride; D015786:Cytochromes b5; D001189:Aryl Hydrocarbon Hydroxylases; D000072467:Cytochrome P450 Family 2; C465241:cytochrome P-450 CYP2B4 (rabbit)