Redox activity of tryptophan residues in recombinant cytochrome c peroxidase and its W51F and W191F mutants

George Tsaprailis, Ann M. English

Research output: Contribution to journalArticle

9 Scopus citations

Abstract

Tryptophan oxidation mediated via the heme was initiated by adding 2, 6, and 20 equivalents of H2O2 to 5 μM recombinant CCP (CCP(MI)) and its W51F and W191F mutants at pH 7.0. Addition of the proteins to 8 M urea (pH 1.5) relieved heme quenching of Trp fluorescence. CCP(MI)-I, W51F-I, and W191F-I, the two-electron oxidized species (FeIV double bond O,R+) formed on addition of 2 equivalents of H2O2, exhibited decreased fluorescence relative to the FeIII forms. Loss of 0.7 Trp in CCP(MI)-I and W51F-I, and 0.2 Trp in W191F-I implies that R.+ is located on Trp 191 in CCP(MI)-I and W51F-I. Spontaneous decay of the FeIV double bond O hemes back to FeIII, followed by reaction with 2 more equivalents of H2O2 after 24 h, resulted in a combined loss of 2.7 (CCP(MI)), 1.5 (W51F), and approx.1 (W191F) Trp. Also, addition of 6 equivalents of H2O2 to the resting FeIII enzymes resulted in loss of approx.2 Trps in CCP(MI) but only approx.1 in W51F and W191F, suggesting that Trp51 becomes redox active in CCP(MI) when >2 equivalents of H2O2 are reduced. Addition of 20 equivalents of H2O2 resulted in a total loss of approx.4, 2.5, and 2 Trp in CCP(MI), W51F, and W191F, respectively. Activity loss largely paralleled Trp loss, and the residual activity of CCP(MI) and W51F exposed to 20 equivalents of H2O2 was 5-19%, while W191F exhibited approx.50% activity. SDS PAGE analysis revealed that oxidized CCP(MI) and W191F were 60-70% monomeric, and W51F 27% monomeric following its reaction with >2 equivalents H2O2. Amino acid analyses confirmed Trp loss and also showed significant Tyr, but not Met, loss in the oxidized proteins. Donors to the heme and pathways of electron migration are proposed based on the combined results.

Original languageEnglish (US)
Pages (from-to)2250-2257
Number of pages8
JournalCanadian Journal of Chemistry
Volume74
Issue number11
DOIs
StatePublished - Jan 1 1996

Keywords

  • HO oxidation
  • Trp mutants
  • cytochrome c peroxidase
  • redox-active residues

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Organic Chemistry

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