Fluorescence investigation of yeast cytochrome c peroxidase oxidation by H2O2 and enzyme activities of the oxidized enzyme

Ted Fox, George Tsaprailis, Ann M. English

Research output: Contribution to journalArticle

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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 58 and 18% H2O2 titer, and 47 and 16% activity, respectively, which shows that radical formation on Trp191 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
StatePublished - 1994
Externally publishedYes

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Cytochrome-c Peroxidase
Enzyme activity
Yeast
Tryptophan
Yeasts
Fluorescence
Oxidation
Enzymes
Titration
Substrates
Cytochromes c
Heme
Dimers
Urea
Quenching
Proteins
Electrons

ASJC Scopus subject areas

  • Biochemistry

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Fluorescence investigation of yeast cytochrome c peroxidase oxidation by H2O2 and enzyme activities of the oxidized enzyme. / Fox, Ted; Tsaprailis, George; English, Ann M.

In: Biochemistry, Vol. 33, No. 1, 1994, p. 186-191.

Research output: Contribution to journalArticle

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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 58 and 18{\%} H2O2 titer, and 47 and 16{\%} activity, respectively, which shows that radical formation on Trp191 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.",
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