Kinetic characterization of Compound I formation in the thermostable cytochrome P450 CYP119

David G. Kellner, Shao Ching Hung, Kara E. Weiss, Stephen G. Sligar

Research output: Contribution to journalArticlepeer-review

Abstract

The kinetics of formation and breakdown of the putative active oxygenating intermediate in cytochrome P450, a ferryl-oxo-(π) porphyrin cation radical (Compound I), have been analyzed in the reaction of a thermostable P450, CYP119, with meta-chloroperoxybenzoic acid (m-CPBA). Upon rapid mixing of m-CPBA with the ferric form of CYP119, an intermediate with spectral features characteristic of a ferryl-oxo-(π) porphyrin cation radical was clearly observed and identified by the absorption maxima at 370, 610, and 690 nm. The rate constant for the formation of Compound I was 3.20 (±0.3) × 105 M-1 s-1 at pH 7.0, 4 °C, and this rate decreased with increasing pH. Compound I of CYP119 decomposed back to the ferric form with a first order rate constant of 29.4 ± 3.4 s-1, which increased with increasing pH. These findings form the first kinetic analysis of Compound I formation and decay in the reaction of m-CPBA with ferric P450.

Original languageEnglish (US)
Pages (from-to)9641-9644
Number of pages4
JournalJournal of Biological Chemistry
Volume277
Issue number12
DOIs
StatePublished - Mar 22 2002

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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