Chemical modification of the histidine residues of purified hepatic cytochrome P-450: influence on substrate binding and the haemoprotein spin state.
A hepatic cytochrome P-450 isolated in an electrophoretically homogeneous form from phenobarbital-treated rats, exists predominantly in the low spin configuration (82% at 20 degrees C). The addition of saturating amounts of the substrate benzphetamine to this haemoprotein shifted the spin equilibrium to the high spin form, resulting in a doubling of the spin equilibrium constant from 0.220 to 0.539 at 20 degrees C. The histidine residues of this low spin, substrate-free cytochrome P-450 were modified in a time- and concentration-dependent manner with diethylpyrocarbonate, and progressive histidine modification resulted in a decrease of both the affinity and extent of substrate interaction with the haemoprotein. Although the histidine-modified haemoprotein maintained the capacity to undergo a temperature-dependent spin transition of the haem iron in the presence of saturating amounts of substrate, this capability was substantially decreased in comparison to the unmodified cytochrome. These results indicate that a histidine residue(s) is involved in the binding of substrate to cytochrome P-450 and hence interferes with the substrate-bound spin equilibrium. Our results further imply that histidine is probably not the sixth ligand of the substrate-free ferric form of the rat liver cytochrome P-450.
D000818:Animals; D001589:Benzphetamine / Q000378:metabolism; D001665:Binding Sites / Q000187:drug effects; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D006639:Histidine / Q000378:metabolism*; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology; D051381:Rats; D011919:Rats, Inbred Strains; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature
D001589:Benzphetamine; D006639:Histidine; D003577:Cytochrome P-450 Enzyme System; D004047:Diethyl Pyrocarbonate