Abstract
Mammalian cytochrome P450 (P450) is a membrane-bound monooxygenase whose catalytic activities require two electrons to be sequentially delivered from its redox partners: cytochrome b5 (cytb5) and cytochrome P450 reductase, both of which are membrane proteins. Although P450 functional activities are known to be affected by lipids, experimental evidence to reveal the effect of membrane on P450-cytb5 interactions is still lacking. Here, we present evidence for the influence of phospholipid bilayers on complex formation between rabbit P450 2B4 (CYP2B4) and rabbit cytb5 at the atomic level, utilizing NMR techniques. General line broadening and modest chemical shift perturbations of cytb5 resonances characterize CYP2B4-cytb5 interactions on the intermediate time scale. More significant intensity attenuation and a more specific protein-protein binding interface are observed in bicelles as compared with lipid-free solution, highlighting the importance of the lipid bilayer in stabilizing stronger and more specific interactions between CYP2B4 and cytb5, which may lead to a more efficient electron transfer. Similar results observed for the interactions between CYP2B4 lacking the transmembrane domain (tr-CYP2B4) and cytb5 imply interactions between tr-CYP2B4 and the membrane surface, which might assist in CYP2B4-cytb5 complex formation by orienting tr-CYP2B4 for efficient contact with cytb5. Furthermore, the observation of weak and nonspecific interactions between CYP2B4 and cytb5 in micelles suggests that lipid bilayer structures and low curvature membrane surface are preferable for CYP2B4-cytb5 complex formation. Results presented in this study provide structural insights into the mechanism behind the important role that the lipid bilayer plays in the interactions between P450s and their redox partners.
Highlights
The functional activities of membrane-bound cytochrome P450s (P450s) are affected by lipids
Interaction between cytb5 and wt-cytochrome P450 2B4 (CYP2B4) —In order to investigate the influence of different membrane mimetic environments on CYP2B4-cytb5 interaction, two-dimensional 15N/1H TROSY-HSQC NMR spectra were recorded to monitor the interaction between 15N-labeled cytb5 and unlabeled wt-CYP2B4 in lipid-free solution, DLPC/DHPC isotropic bicelles, and DPC micelles
Effect of DLPC/DHPC Isotropic Bicelles on CYP2B4-cytb5 Interaction—The importance of phospholipids in the cytochrome P450 reconstituted system has been known since the first successful reconstitution of cytochrome P450-dependent activities [13,14,15,16, 57, 58]
Summary
The functional activities of membrane-bound cytochrome P450s (P450s) are affected by lipids. Results: Stronger interactions between P450 2B4 (CYP2B4) and cytochrome b5 (cytb5) are observed in bicelles as compared with lipid-free solution or micelles. Mammalian cytochrome P450 (P450) is a membrane-bound monooxygenase whose catalytic activities require two electrons to be sequentially delivered from its redox partners: cytochrome b5 (cytb5) and cytochrome P450 reductase, both of which are membrane proteins. More significant intensity attenuation and a more specific protein-protein binding interface are observed in bicelles as compared with lipid-free solution, highlighting the importance of the lipid bilayer in stabilizing stronger and more specific interactions between CYP2B4 and cytb, which may lead to a more efficient electron transfer. Results presented in this study provide structural insights into the mechanism behind the important role that the lipid bilayer plays in the interactions between P450s and their redox partners.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
