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. 1970 Sep;67(1):172–179. doi: 10.1073/pnas.67.1.172

Electron Paramagnetic Resonance Study of the High- and Low-Spin Forms of Cytochrome P-450 in Liver and in Liver Microsomes from a Methylcholanthrene-Treated Rabbit*

J Peisach 1,2,3,†, W E Blumberg 1,2,3
PMCID: PMC283184  PMID: 4318775

Abstract

The high- and low-spin forms of cytochrome P-450 were observed by electron paramagnetic resonance (epr) in a liver slice and in hepatic microsomes from a rabbit injected with methylcholanthrene. Quantitation of the epr absorption of these two chemical species and comparison with a preparation from a control rabbit showed that the single injection of drug increased the concentration of cytochrome P-450 to more than ten times its control value. Analysis of the epr spectrum for the high-spin compound showed that it is the most rhombically distorted ferric heme iron site yet observed (E/D = 0.10). It is suggested that microsomal cytochrome P-450 can exist in two interconvertible forms, in which the heme iron can either be high-spin or low-spin, depending upon the nature of the nonporphyrin ligands of the metal. There seems to be no need to postulate the existence of two different cytochrome P-450 proteins.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. BEINERT H., HEINEN W., PALMER G. Applications of combined low temperature optical and electron paramagnetic resonance spectroscopy to the study of oxidative enzymes. Brookhaven Symp Biol. 1962 Dec;15:229–265. [PubMed] [Google Scholar]
  2. Bois-Poltoratsky R., Ehrenberg A. Magnetic and spectrophotometric investigations of cytochrome b5. Eur J Biochem. 1967 Oct;2(3):361–365. doi: 10.1111/j.1432-1033.1967.tb00146.x. [DOI] [PubMed] [Google Scholar]
  3. Cammer W., Schenkman J. B., Estabrook R. W. EPR measurements of substrate interaction with cytochrome P-450. Biochem Biophys Res Commun. 1966 May 3;23(3):264–268. doi: 10.1016/0006-291x(66)90539-0. [DOI] [PubMed] [Google Scholar]
  4. Ernster L., Orrenius S. Substrate-induced synthesis of the hydroxylating enzyme system of liver microsomes. Fed Proc. 1965 Sep-Oct;24(5):1190–1199. [PubMed] [Google Scholar]
  5. GARFINKEL D. Studies on pig liver microsomes. I. Enzymic and pigment composition of different microsomal fractions. Arch Biochem Biophys. 1958 Oct;77(2):493–509. doi: 10.1016/0003-9861(58)90095-x. [DOI] [PubMed] [Google Scholar]
  6. Gnosspelius Y., Thor H., Orrenius S. A comparative study on the effects of phenobarbital and 3,4-benzpyrene on the hydroxylating enzyme system of rat-liver microsomes. Chem Biol Interact. 1969 Dec;1(2):125–137. doi: 10.1016/0009-2797(69)90001-5. [DOI] [PubMed] [Google Scholar]
  7. Gunsalus I. C. A soluble methylene hydroxylase system: structure and role of cytochrome P-450 and iron-sulfur protein components. Hoppe Seylers Z Physiol Chem. 1968 Nov;349(11):1610–1613. [PubMed] [Google Scholar]
  8. Imai Y., Sato R. Substrate interaction with hydroxylase system in liver microsomes. Biochem Biophys Res Commun. 1966 Mar 22;22(6):620–626. doi: 10.1016/0006-291x(66)90191-4. [DOI] [PubMed] [Google Scholar]
  9. KLINGENBERG M. Pigments of rat liver microsomes. Arch Biochem Biophys. 1958 Jun;75(2):376–386. doi: 10.1016/0003-9861(58)90436-3. [DOI] [PubMed] [Google Scholar]
  10. Katagiri M., Ganguli B. N., Gunsalus I. C. A soluble cytochrome P-450 functional in methylene hydroxylation. J Biol Chem. 1968 Jun 25;243(12):3543–3546. [PubMed] [Google Scholar]
  11. Lu A. Y., Junk K. W., Coon M. J. Resolution of the cytochrome P-450-containing omega-hydroxylation system of liver microsomes into three components. J Biol Chem. 1969 Jul 10;244(13):3714–3721. [PubMed] [Google Scholar]
  12. Mason H. S., North J. C., Vanneste M. Microsomal mixed-function oxidations: the metabolism of xenobiotics. Fed Proc. 1965 Sep-Oct;24(5):1172–1180. [PubMed] [Google Scholar]
  13. Mitani F., Horie S. Studies on P-450. VI. The spin state of P-450 solubilized from bovine adrenocortical mitochondria. J Biochem. 1969 Aug;66(2):139–149. doi: 10.1093/oxfordjournals.jbchem.a129129. [DOI] [PubMed] [Google Scholar]
  14. Murakami K., Mason H. S. An electron spin resonance study of microsomal Fe. J Biol Chem. 1967 Mar 25;242(6):1102–1110. [PubMed] [Google Scholar]
  15. OMURA T., SATO R. THE CARBON MONOXIDE-BINDING PIGMENT OF LIVER MICROSOMES. I. EVIDENCE FOR ITS HEMOPROTEIN NATURE. J Biol Chem. 1964 Jul;239:2370–2378. [PubMed] [Google Scholar]
  16. Omura T., Sato R., Cooper D. Y., Rosenthal O., Estabrook R. W. Function of cytochrome P-450 of microsomes. Fed Proc. 1965 Sep-Oct;24(5):1181–1189. [PubMed] [Google Scholar]
  17. Peterson J. A., Coon M. J. Enzymatic omega-oxidation. 3. Purification and properties of rubredoxin, a component of the omega-hydroxylation system of Pseudomonas oleovorans. J Biol Chem. 1968 Jan 25;243(2):329–334. [PubMed] [Google Scholar]
  18. Remmer H., Schenkman J., Estabrook R. W., Sasame H., Gillette J., Narasimhulu S., Cooper D. Y., Rosenthal O. Drug interaction with hepatic microsomal cytochrome. Mol Pharmacol. 1966 Mar;2(2):187–190. [PubMed] [Google Scholar]
  19. Salmeen I., Palmer G. Electron paramagnetic resonance of beef-heart ferricytochrome c. J Chem Phys. 1968 Mar 1;48(5):2049–2052. doi: 10.1063/1.1669014. [DOI] [PubMed] [Google Scholar]
  20. Schenkman J. B., Greim H., Zange M., Remmer H. On the problem of possible other forms of cytochrome P450 in liver microsomes. Biochim Biophys Acta. 1969 Jan 7;171(1):23–31. doi: 10.1016/0005-2744(69)90102-8. [DOI] [PubMed] [Google Scholar]
  21. Sladek N. E., Mannering G. J. Evidence for a new P-450 hemoprotein in hepatic microsomes from methylcholanthrene treated rats. Biochem Biophys Res Commun. 1966 Sep 8;24(5):668–674. doi: 10.1016/0006-291x(66)90376-7. [DOI] [PubMed] [Google Scholar]
  22. Sladek N. E., Mannering G. J. Induction of drug metabolism. I. Differences in the mechanisms by which polycyclic hydrocarbons and phenobarbital produce their inductive effects on microsomal N-demethylating systems. Mol Pharmacol. 1969 Mar;5(2):174–185. [PubMed] [Google Scholar]
  23. Sladek N. E., Mannering G. J. Induction of drug metabolism. II. Qualitative differences in the microsomal N-demethylating systems stimulated by polycyclic hydrocarbons and by phenobarbital. Mol Pharmacol. 1969 Mar;5(2):186–199. [PubMed] [Google Scholar]
  24. Tsai R., Yu C. A., Gunsalus I. C., Peisach J., Blumberg W., Orme-Johnson W. H., Beinert H. Spin-state changes in cytochrome P-450cam on binding of specific substrates. Proc Natl Acad Sci U S A. 1970 Aug;66(4):1157–1163. doi: 10.1073/pnas.66.4.1157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Van Gelder B. F., Beinert H. Studies of the heme components of cytochrome c oxidase by EPR spectroscopy. Biochim Biophys Acta. 1969 Sep 16;189(1):1–24. doi: 10.1016/0005-2728(69)90219-9. [DOI] [PubMed] [Google Scholar]
  26. Whysner J. A., Ramseyer J., Kazmi G. M., Harding B. W. Substrate induced spin state changes in cytochrome P-450. Biochem Biophys Res Commun. 1969 Aug 22;36(5):795–801. doi: 10.1016/0006-291x(69)90679-2. [DOI] [PubMed] [Google Scholar]

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