On the function of cytochrome b5 in the cytochrome P-450-dependent oxygenase system.
Complex formation between the phenobarbital-inducible form of rabbit liver microsomal cytochrome P-450 incorporated into phosphatidylcholine and detergent-solubilized cytochrome b5 is associated with a low-to-high spin transition of the former pigment. It is concluded that the proteins combine in a 1:1 molar ratio. CD spectral analysis in the far uv region reveals that interaction of the cytochromes results in a conformational change of one or both hemoproteins. Such a cytochrome b5-induced structural alteration of the reconstituted enzyme system is accompanied by an increase in affinity of 4-chloroaniline for cytochrome P-450, as measured in terms of cumene hydroperoxide-supported N-oxidation of the arylamine; the maximum velocity of the catalytic process remains unchanged. Similarly, incorporation into the assay media of cytochrome b5 decreases the apparent Kd values of both the amine substrate and the oxygen donor, as determined by optical titration. Stopped-flow spectrophotometric studies on the influence of cytochrome b5 on the kinetics of binding to cytochrome P-450 of 4-chloroaniline and/or cumene hydroperoxide show that the rates of formation and decay of the adducts change as the molar ratio of cytochrome b5 to cytochrome P-450 varies. Moreover, cytochrome b5 modifies the activation energies required for production of the substrate-bound oxy complex. These findings suggest that cytochrome b5, apart from its well-known role as an electron carrier, might exert an effector function in the cytochrome P-450 system.
D000814:Aniline Compounds / Q000378:metabolism; D000818:Animals; D001555:Benzene Derivatives / Q000378:metabolism; D002384:Catalysis; D003577:Cytochrome P-450 Enzyme System; D003573:Cytochrome b Group / Q000502:physiology*; D015786:Cytochromes b5; D004735:Energy Transfer; D005260:Female; D066298:In Vitro Techniques; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D010105:Oxygenases / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism; D011817:Rabbits; D013696:Temperature
D000814:Aniline Compounds; D001555:Benzene Derivatives; D003573:Cytochrome b Group; D010743:Phospholipids; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D010105:Oxygenases; C007164:cumene hydroperoxide; C004658:4-chloroaniline