Motional dynamics of a spin labeled substrate analogue bound to cytochrome P-450: saturation transfer EPR studies.
The rotational motion of the spin labeled substrate analogue n- propylisocyanide bound to the active center of cytochrome P-450 was studied by saturation transfer EPR. The observed motional rate characterized by an effective rotational correlation time tau R of about 40 ns at 20 degrees C is at least 3 orders of magnitude higher than the macromolecular rotational diffusion of cytochrome P-450 in the microsomal membrane and represents a considerable motion in relation to the whole enzyme molecule. The tau R value is independent on the degree of purification of the enzyme system as revealed by measurements of (1) liver microsomes, (2) partially purified cytochrome P-450, and (3) cytochrome P-450 LM2 but shows a characteristic temperature dependence in the case of microsomes resulting in breaks in the Arrhenius plot at temperatures which correspond to phase transitions of the phospholipids. The results indicate that the mobility of the bound substrate analogue reflecting a relatively high conformational flexibility of the substrate binding region which depends on the state of the lipids and can therefore be influenced by them. These results support the assumption that cytochrome P-450 is capable of forming manifold binding to substrate molecules differing in stereochemical structures because of the conformational flexibility of its binding region.
D000818:Animals; D001665:Binding Sites; D003497:Cyclic N-Oxides / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D008055:Lipids; D008297:Male; D010634:Phenobarbital / Q000494:pharmacology; D011487:Protein Conformation; D011817:Rabbits; D013113:Spin Labels; D013237:Stereoisomerism; D013696:Temperature
D003497:Cyclic N-Oxides; D008055:Lipids; D013113:Spin Labels; C041909:4-(1-oxo-2,2,6,6-tetramethylpiperidyl)-3-isocyano-n-propyl ether; D003577:Cytochrome P-450 Enzyme System; D010634:Phenobarbital