Fluorescence Investigation of Yeast Cytochrome c Peroxidase Oxidation by H2O2 and Enzyme Activities of the Oxidized Enzyme

Ted Fox, George Tsaprailis, Ann M. English

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24 Scopus citations

Abstract

The role of tryptophan residues as endogenous electron donors in cytochrome c peroxidase (CCP) was examined by protein steady-state fluorescence. Compound I and more highly oxidized forms of CCP were formed by adding 1,3, and 10 equiv of H2O2 to 5 µM protein at pH 7.0 in the absence of exogenous reducing substrates. Addition of native CCP to 8 M urea at pH 1.5 relieved heme quenching, and compound I exhibited 90 ± 4% fluorescence relative to unoxidized CCP, consistent with the loss of 0.7 ± 0.2 tryptophan and the assignment of the primary radical site to Trp191. CCP oxidized with 10-fold excess H2O2 exhibited 65 ± 1% relative fluorescence, indicating loss of 2.4 ± 0.1 tryptophans. Compound I and the higher oxidized forms of CCP spontaneously decayed to ferric CCP species over ∼24 h with the loss of ∼0.5 additional tryptophan in each case. The 24-h decay product of compound I exhibited 73% activity, 74% H2O2 titer, and titration led to the further oxidation of ∼0.6 tryptophan. However, no further tryptophan oxidation was observed on titration of the 24-h decay products of samples initially oxidized with 3 and 10 equiv of H2O2. These samples exhibited 5 8 and 18% H2O2 titer, and 47 and 16% activity, respectively, which shows that radical formation on Trpl91 is not required for activity. The fluorescence decrease with time paralleled the decrease in activity of H2O2-oxidized CCP using both ferrocytochrome c and ferrocyanide as substrates, indicating that tryptophan and activity loss occurred on similar time scales. Since CCP reduced 3 and 10 equiv of H2O2 within 5 and 20 min, respectively, but fluorescence and activity loss increased slightly over 24 h, charge migration must occur in the polypeptide during decay, giving rise to 7–33% dimer formation along with the loss of the 0.5 additional tryptophan.

Original languageEnglish (US)
Pages (from-to)186-191
Number of pages6
JournalBiochemistry
Volume33
Issue number1
DOIs
StatePublished - Jan 1 1994
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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