Fluorescence investigation of yeast cytochrome c peroxidase oxidation by H2O2 and enzyme activities of the oxidized enzyme.
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 microM 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 approximately 24 h with the loss of approximately 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 approximately 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 of 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.(ABSTRACT TRUNCATED AT 250 WORDS)
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Department of Chemistry and Biochemistry, Concordia University, Montreal, Quebec, Canada.
Tsaprailis
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English
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D000595:Amino Acid Sequence; D003578:Cytochrome-c Peroxidase / Q000737:chemistry* / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism*; D007700:Kinetics; D010084:Oxidation-Reduction; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013050:Spectrometry, Fluorescence / Q000379:methods; D013053:Spectrophotometry; D014364:Tryptophan
D014364:Tryptophan; D006861:Hydrogen Peroxide; D003578:Cytochrome-c Peroxidase