Histidine residues in rabbit liver microsomal cytochrome P-450 2B4 control electron transfer from NADPH-cytochrome P-450 reductase and cytochrome b5.
Treatment of cytochrome P-450 2B4 (P-450 2B4) with diethylpyrocarbonate to introduce 10-11 equivalents of acylating agent per polypeptide chain resulted in the selective derivatization of histidine residues characterized by differential susceptibility toward the modifier. Second-derivative spectral analysis as well as fluorescence measurements disproved gross alterations in P-450 2B4 structure as a consequence of labelling. The modified haemoprotein retained its ability to bind hexobarbital and catalyse cumene hydroperoxide-sustained N-demethylation of the barbiturate. However, there was a steady attenuation of NAD(P)H-driven electron flux with increasing extent of P-450 2B4 carbethoxylation in reconstituted systems fortified with either NADPH-cytochrome P-450 reductase or NADH-cytochrome b5 reductase/cytochrome b5 as the redox partners, with 50% inhibition occurring when 6-7 histidines were blocked. Hampered P-450 2B4 reductase activities recovered to differing degrees upon treatment of the acylated mono-oxygenase with neutral hydroxylamine. Spectral data indicated that docking of the redox components to derivatized P-450 2B4 was not perturbed, so that disruption of the electron flows most likely resulted from some injury of the electron-transfer mechanisms.
Hlavica
P
P
Walther-Straub-Institut für Pharmakologie und Toxikologie, Universität München, Germany.
Lehnerer
M
M
Eulitz
M
M
D000215:Acylation; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001665:Binding Sites; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism*; D015786:Cytochromes b5 / Q000378:metabolism*; D004047:Diethyl Pyrocarbonate; D004579:Electron Transport; D006639:Histidine / Q000737:chemistry; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D015394:Molecular Structure; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*; D010084:Oxidation-Reduction; D011817:Rabbits; D013250:Steroid Hydroxylases / Q000737:chemistry* / Q000378:metabolism*
D009243:NAD; D006639:Histidine; D009249:NADP; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C050356:steroid 15-alpha-hydroxylase; D009251:NADPH-Ferrihemoprotein Reductase; D004047:Diethyl Pyrocarbonate