Mesopone cytochrome c peroxidase

Functional model of heme oxygenated oxidases

Chad E. Immoos, B. Bhaskar, Michael Cohen, Tiffany P. Barrows, Patrick J. Farmer, T. L. Poulos

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

The effect of heme ring oxygenation on enzyme structure and function has been examined in a reconstituted cytochrome c peroxidase. Oxochlorin derivatives were formed by OsO4 treatment of mesoporphyrin followed by acid-catalyzed pinacol rearrangement. The northern oxochlorin isomers were isolated by chromatography, and the regio-isomers assignments determined by 2D COSY and NOE 1H NMR. The major isomer, 4-mesoporphyrinone (Mp), was metallated with FeCl2 and reconstituted into cytochrome c peroxidase (CcP) forming a hybrid green protein, MpCcP. The heme-altered enzyme has 99% wild-type peroxidase activity with cytochrome c. EPR spectroscopy of MpCcP intermediate compound I verifies the formation of the Trp191 radical similar to wild-type CcP in the reaction cycle. Peroxidase activity with small molecules is varied: guaiacol turnover increases approximately five-fold while that with ferrocyanide is ∼85% of native. The electron-withdrawing oxo-substitutents on the cofactor cause a ∼60-mV increase in FeIII/FeII reduction potential. The present investigation represents the first structural characterization of an oxochlorin protein with X-ray intensity data collected to 1.70 Å. Although a mixture of R- and S-mesopone isomers of the FeMP cofactor was used during heme incorporation into the apo-protein, only the S-isomer is found in the crystallized protein.

Original languageEnglish (US)
Pages (from-to)635-643
Number of pages9
JournalJournal of Inorganic Biochemistry
Volume91
Issue number4
DOIs
StatePublished - Sep 20 2002
Externally publishedYes

Fingerprint

Cytochrome-c Peroxidase
Heme
Isomers
Oxidoreductases
Peroxidase
Guaiacol
Proteins
Protein S
Enzymes
Cytochromes c
Chromatography
Spectrum Analysis
X-Rays
Oxygenation
Electrons
Acids
Paramagnetic resonance
Nuclear magnetic resonance
Spectroscopy
Derivatives

Keywords

  • Cytochrome c peroxidase
  • Heme cd model
  • Reconstituted oxochlorin

ASJC Scopus subject areas

  • Biochemistry
  • Inorganic Chemistry

Cite this

Mesopone cytochrome c peroxidase : Functional model of heme oxygenated oxidases. / Immoos, Chad E.; Bhaskar, B.; Cohen, Michael; Barrows, Tiffany P.; Farmer, Patrick J.; Poulos, T. L.

In: Journal of Inorganic Biochemistry, Vol. 91, No. 4, 20.09.2002, p. 635-643.

Research output: Contribution to journalArticle

Immoos, Chad E. ; Bhaskar, B. ; Cohen, Michael ; Barrows, Tiffany P. ; Farmer, Patrick J. ; Poulos, T. L. / Mesopone cytochrome c peroxidase : Functional model of heme oxygenated oxidases. In: Journal of Inorganic Biochemistry. 2002 ; Vol. 91, No. 4. pp. 635-643.
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AB - The effect of heme ring oxygenation on enzyme structure and function has been examined in a reconstituted cytochrome c peroxidase. Oxochlorin derivatives were formed by OsO4 treatment of mesoporphyrin followed by acid-catalyzed pinacol rearrangement. The northern oxochlorin isomers were isolated by chromatography, and the regio-isomers assignments determined by 2D COSY and NOE 1H NMR. The major isomer, 4-mesoporphyrinone (Mp), was metallated with FeCl2 and reconstituted into cytochrome c peroxidase (CcP) forming a hybrid green protein, MpCcP. The heme-altered enzyme has 99% wild-type peroxidase activity with cytochrome c. EPR spectroscopy of MpCcP intermediate compound I verifies the formation of the Trp191 radical similar to wild-type CcP in the reaction cycle. Peroxidase activity with small molecules is varied: guaiacol turnover increases approximately five-fold while that with ferrocyanide is ∼85% of native. The electron-withdrawing oxo-substitutents on the cofactor cause a ∼60-mV increase in FeIII/FeII reduction potential. The present investigation represents the first structural characterization of an oxochlorin protein with X-ray intensity data collected to 1.70 Å. Although a mixture of R- and S-mesopone isomers of the FeMP cofactor was used during heme incorporation into the apo-protein, only the S-isomer is found in the crystallized protein.

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