Detection of a tryptophan radical as an intermediate species in the reaction of horseradish peroxidase mutant (phe-221 → trp) and hydrogen peroxide

Atsushi Morimoto, Motomasa Tanaka, Satoshi Takahashi, Koichiro Ishimori, Hiroshi Hori, Isao Morishima

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44 Citations (Scopus)

Abstract

The crucial reaction intermediate in the reaction of peroxidase with hydrogen peroxide (H2O2), compound I, contains a porphyrin π-cation radical in horseradish peroxidase (HRP), which catalyzes oxidation of small organic and inorganic compounds, whereas cytochrome c peroxidase (CcP) has a radical center on the tryptophan residue (Trp.191) and oxidizes the redox partner, cytochrome c. To investigate the roles of the amino acid residue near the heme active center in discriminating the function of the peroxidases in these two enzymes, we prepared a CcP-like HRP mutant, F221W (Phe-221 → Trp). Although the rapid spectral scanning and stoppedflow experiments confirmed that the F221W mutant reacts with H2O2 to form the porphyrin π- cation radical at the same rate as for the wild-type enzyme, the characteristic spectral features of the porphyrin π-cation radical disappeared rapidly, and were converted to the compound II-type spectrum. The EPR spectrum of the resultant species produced by reduction of the porphytin π-cation radical, however, was quite different from that of compound H in HRP, showing typical signals from a Trp radical as found for CcP. The sequential radical formation from the porphyrin ring to the Trp residue implies that the proximal Trp is a key residue in the process of the radical transfer from the porphyrin ring, which differentiates the function of peroxidases.

Original languageEnglish
Pages (from-to)14753-14760
Number of pages8
JournalJournal of Biological Chemistry
Volume273
Issue number24
DOIs
Publication statusPublished - 1998 Jun 12
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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