Histidine residues in rabbit liver microsomal cytochrome P -450 2B4 control electron transfer from NADPH-cytochrome P -450 reductase and cytochrome b 5

• Published in: Biochemical journal 1996-09, Vol.318 (3), p.857-862
• Main Authors: HLAVICA, Peter, LEHNERER, Michael, EULITZ, Manfred
• Format: Article
• Language: English
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj3180857

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.