Histidine residues in rabbit liver microsomal cytochrome P-450 2B4 control electron transfer from NADPH-cytochrome P-450 reductase and cytochrome b5

摘要

pTreatment of cytochrome iP/i-450 2B4 (iP/i-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 iP/i-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 iP/i-450 2B4 carbethoxylation in reconstituted systems fortified with either NADPH-cytochrome iP/i-450 reductase or NADH-cytochrome ib/isub5/sub reductase/cytochrome ib/isub5/sub as the redox partners, with 50% inhibition occurring when 6–7 histidines were blocked. Hampered iP/i-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 iP/i-450 2B4 was not perturbed, so that disruption of the electron flows most likely resulted from some injury of the electron-transfer mechanisms./p