Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1989 Oct;86(20):7823–7827. doi: 10.1073/pnas.86.20.7823

Uncoupling of the cytochrome P-450cam monooxygenase reaction by a single mutation, threonine-252 to alanine or valine: possible role of the hydroxy amino acid in oxygen activation.

M Imai 1, H Shimada 1, Y Watanabe 1, Y Matsushima-Hibiya 1, R Makino 1, H Koga 1, T Horiuchi 1, Y Ishimura 1
PMCID: PMC298163  PMID: 2510153

Abstract

Site-directed mutants of cytochrome P-450cam (the cytochrome P-450 that acts as the terminal monooxygenase in the d-camphor monooxygenase system), in which threonine-252 had been changed to alanine, valine, or serine, were employed to study the role of the hydroxy amino acid in the monooxygenase reaction. The mutant enzymes were expressed in Escherichia coli and were purified by a conventional method. All the mutant enzymes in the presence of d-camphor exhibited optical absorption spectra almost indistinguishable from those of the wild-type enzyme in their ferric, ferrous, oxygenated, and carbon monoxide ferrous forms. In a reconstituted system with putidaredoxin and its reductase, the alanine enzyme consumed O2 at a rate (1100 per min per heme) comparable to that of the wild-type enzyme (1330 per min per heme), whereas the amount of exo-5-hydroxycamphor formed was less than 10% of that formed by the wild-type enzyme. About 85% of the O2 consumed was recovered as H2O2. The valine enzyme also exhibited an oxidase activity to yield H2O2 accompanied by a relative decrease in the monooxygenase activity. On the other hand, the serine enzyme exhibited essentially the same monooxygenase activity as that of the wild-type enzyme. Thus, uncoupling of O2 consumption from the monooxygenase function was produced by the substitution of an amino acid without a hydroxyl group. When binding of O2 to the ferrous forms was examined, the alanine and valine enzymes formed instantaneously an oxygenated form, which slowly decomposed to the ferric form with rates of 5.5 and 3.2 x 10(-3) sec-1 for the former and latter enzymes, respectively. Since these rates were too slow to account for the overall rates of O2 consumption, the formation of H2O2 was considered to proceed not by way of this route but through the decomposition of a peroxide complex formed by reduction of the oxygenated form by reduced putidaredoxin. Based on these findings, a possible mechanism for oxygen activation in this monooxygenase reaction has been discussed.

Full text

PDF
7823

Selected References

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

  1. Atkins W. M., Sligar S. G. The roles of active site hydrogen bonding in cytochrome P-450cam as revealed by site-directed mutagenesis. J Biol Chem. 1988 Dec 15;263(35):18842–18849. [PubMed] [Google Scholar]
  2. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cohen S. N., Chang A. C., Hsu L. Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci U S A. 1972 Aug;69(8):2110–2114. doi: 10.1073/pnas.69.8.2110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Estabrook R. W., Hildebrandt A. G., Baron J., Netter K. J., Leibman K. A new spectral intermediate associated with cytochrome P-450 function in liver microsomes. Biochem Biophys Res Commun. 1971 Jan 8;42(1):132–139. doi: 10.1016/0006-291x(71)90372-x. [DOI] [PubMed] [Google Scholar]
  5. Guengerich F. P., Ballou D. P., Coon M. J. Spectral intermediates in the reaction of oxygen with purified liver microsomal cytochrome P-450. Biochem Biophys Res Commun. 1976 Jun 7;70(3):951–956. doi: 10.1016/0006-291x(76)90684-7. [DOI] [PubMed] [Google Scholar]
  6. Guengerich F. P. Oxidation-reduction properties of rat liver cytochromes P-450 and NADPH-cytochrome p-450 reductase related to catalysis in reconstituted systems. Biochemistry. 1983 Jun 7;22(12):2811–2820. doi: 10.1021/bi00281a007. [DOI] [PubMed] [Google Scholar]
  7. Gunsalus I. C., Wagner G. C. Bacterial P-450cam methylene monooxygenase components: cytochrome m, putidaredoxin, and putidaredoxin reductase. Methods Enzymol. 1978;52:166–188. doi: 10.1016/s0076-6879(78)52019-3. [DOI] [PubMed] [Google Scholar]
  8. Hager L. P., Doubek D. L., Silverstein R. M., Hargis J. H., Martin J. C. Chloroperoxidase. IX. The structure of compound I. J Am Chem Soc. 1972 Jun 14;94(12):4364–4366. doi: 10.1021/ja00767a068. [DOI] [PubMed] [Google Scholar]
  9. Ishimura Y., Ullrich V., Peterson J. A. Oxygenated cytochrome P-450 and its possible role in enzymic hydroxylation. Biochem Biophys Res Commun. 1971 Jan 8;42(1):140–146. doi: 10.1016/0006-291x(71)90373-1. [DOI] [PubMed] [Google Scholar]
  10. Koga H., Rauchfuss B., Gunsalus I. C. P450cam gene cloning and expression in Pseudomonas putida and Escherichia coli. Biochem Biophys Res Commun. 1985 Jul 16;130(1):412–417. doi: 10.1016/0006-291x(85)90432-2. [DOI] [PubMed] [Google Scholar]
  11. Kramer W., Drutsa V., Jansen H. W., Kramer B., Pflugfelder M., Fritz H. J. The gapped duplex DNA approach to oligonucleotide-directed mutation construction. Nucleic Acids Res. 1984 Dec 21;12(24):9441–9456. doi: 10.1093/nar/12.24.9441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Makino R., Tanaka T., Iizuka T., Ishimura Y., Kanegasaki S. Stoichiometric conversion of oxygen to superoxide anion during the respiratory burst in neutrophils. Direct evidence by a new method for measurement of superoxide anion with diacetyldeuteroheme-substituted horseradish peroxidase. J Biol Chem. 1986 Sep 5;261(25):11444–11447. [PubMed] [Google Scholar]
  13. OMURA T., SATO R. A new cytochrome in liver microsomes. J Biol Chem. 1962 Apr;237:1375–1376. [PubMed] [Google Scholar]
  14. Poulos T. L., Finzel B. C., Gunsalus I. C., Wagner G. C., Kraut J. The 2.6-A crystal structure of Pseudomonas putida cytochrome P-450. J Biol Chem. 1985 Dec 25;260(30):16122–16130. [PubMed] [Google Scholar]
  15. Poulos T. L., Finzel B. C., Howard A. J. Crystal structure of substrate-free Pseudomonas putida cytochrome P-450. Biochemistry. 1986 Sep 9;25(18):5314–5322. doi: 10.1021/bi00366a049. [DOI] [PubMed] [Google Scholar]
  16. Poulos T. L., Finzel B. C., Howard A. J. High-resolution crystal structure of cytochrome P450cam. J Mol Biol. 1987 Jun 5;195(3):687–700. doi: 10.1016/0022-2836(87)90190-2. [DOI] [PubMed] [Google Scholar]
  17. Poulos T. L., Kraut J. The stereochemistry of peroxidase catalysis. J Biol Chem. 1980 Sep 10;255(17):8199–8205. [PubMed] [Google Scholar]
  18. Sanger F. Determination of nucleotide sequences in DNA. Science. 1981 Dec 11;214(4526):1205–1210. doi: 10.1126/science.7302589. [DOI] [PubMed] [Google Scholar]
  19. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Schonbaum G. R., Lo S. Interaction of peroxidases with aromatic peracids and alkyl peroxides. Product analysis. J Biol Chem. 1972 May 25;247(10):3353–3360. [PubMed] [Google Scholar]
  21. Sligar S. G., Lipscomb J. D., Debrunner P. G., Gunsalus I. C. Superoxide anion production by the autoxidation of cytochrome P450cam. Biochem Biophys Res Commun. 1974 Nov 6;61(1):290–296. doi: 10.1016/0006-291x(74)90565-8. [DOI] [PubMed] [Google Scholar]
  22. Tyson C. A., Lipscomb J. D., Gunsalus I. C. The role of putidaredoxin and P450 cam in methylene hydroxylation. J Biol Chem. 1972 Sep 25;247(18):5777–5784. [PubMed] [Google Scholar]
  23. Unger B. P., Gunsalus I. C., Sligar S. G. Nucleotide sequence of the Pseudomonas putida cytochrome P-450cam gene and its expression in Escherichia coli. J Biol Chem. 1986 Jan 25;261(3):1158–1163. [PubMed] [Google Scholar]
  24. White R. E., Coon M. J. Oxygen activation by cytochrome P-450. Annu Rev Biochem. 1980;49:315–356. doi: 10.1146/annurev.bi.49.070180.001531. [DOI] [PubMed] [Google Scholar]
  25. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES