Studies on hydroperoxide-dependent substrate hydroxylation by purified liver microsomal cytochrome P-450

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Abstract

Highly purified liver microsomal cytochrome P-450 catalyzes the hydroperoxide-dependent hydroxylation of a variety of substrates in the absence of NADPH, NADPH-cytochrome P-450 reductase, and molecular oxygen. The addition of phosphatidylcholine is necessary for maximal activity. The absence of flavoproteins and cytochrome b5 from the cytochrome P-450 preparations rules out the involvement of other known microsomal electron carriers. The ferrous form of cytochrome P-450 is not involved in peroxide-dependent hydroxylation reactions, as indicated by the lack of inhibition by carbon monoxide. With cumene hydroperoxide present, a variety of substrates is attacked, including N-methylaniline, N,N-dimethylaniline, cyclohexane, benzphetamine, and aminopyrine. With benzphetamine as the substrate, cumene hydroperoxide may be replaced by other peroxides, including hydrogen peroxide, or by peracids or sodium chlorite. A study of the stoichiometry indicated that equimolar amounts of N-methylaniline, formaldehyde, and cumyl alcohol (α,α-dimethylbenzyl alcohol) are formed in the reaction of N,N-dimethylaniline with cumene hydroperoxide. Since H218O is incorporated only slightly into cyclohexanol in the reaction of cyclohexane with cumene hydroperoxide, it appears that the oxygen atom in cyclohexanol is derived primarily from the peroxide. The data obtained are in accord with a peroxidase-like mechanism for the action of cytochrome P-450.

References (48)

  • A.Y.H. Lu et al.

    J. Biol. Chem

    (1968)
  • M.J. Coon et al.
  • A.Y.H. Lu et al.

    J. Biol. Chem

    (1969)
  • H.W. Strobel et al.

    J. Biol. Chem

    (1970)
  • T.A. van der Hoeven et al.

    J. Biol. Chem

    (1974)
  • T.A. van der Hoeven et al.

    Biochem. Biophys. Res. Commun

    (1974)
  • D.A. Haugen et al.

    J. Biol. Chem

    (1975)
  • D.P. Ballou et al.

    FEBS Lett

    (1974)
  • F.P. Guengerich et al.

    J. Biol. Chem

    (1975)
  • R.W. Estabrook et al.

    Biochem. Biophys. Res. Commun

    (1971)
  • I.C. Gunsalus et al.

    Chem. Biol. Interact

    (1971)
  • Y. Ishimura et al.

    Biochem. Biophys. Res. Commun

    (1971)
  • H.W. Strobel et al.

    J. Biol. Chem

    (1971)
  • C. Chen et al.

    Biochim. Biophys. Acta

    (1968)
  • C. Chen et al.

    Biochim. Biophys. Acta

    (1969)
  • J.E. Van Lier et al.

    Biochem. Biophys. Res. Commun

    (1970)
  • F.F. Kadlubar et al.

    Biochem. Biophys. Res. Commun

    (1973)
  • A.D. Rahimtula et al.

    Biochem. Biophys. Res. Commun

    (1974)
  • Å. Ellin et al.

    FEBS Lett

    (1975)
  • A.D. Rahimtula et al.

    Biochem. Biophys. Res. Commun

    (1975)
  • E.G. Hrycay et al.

    FEBS Lett

    (1975)
  • E.G. Hrycay et al.

    Biochem. Biophys. Res. Commun

    (1975)
  • A.Y.H. Lu et al.

    Biochem. Biophys. Res. Commun

    (1969)
  • T. Omura et al.

    J. Biol. Chem

    (1964)
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    This research was supported by Grant BMS71-01195 from the National Science Foundation and Grant AM-10339 from the United States Public Health Service. Preliminary reports of part of this investigation have been presented (1, 2).

    2

    Postdoctoral Fellow, United States Public Health Service.

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