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

Abstract

Tryptophan oxidation mediated via the heme was initiated by adding 2, 6, and 20 equivalents of H20, to 5 p,M recombinant CCP (CCP(M1)) and its W5 IF 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(M1)-I, W5 1F-I, and W 191F-I, the two-electron oxidized species (F~'~=O,R'+) formed on addition of 2 equivalents of H,02, exhibited decreased fluorescence relative to the ~e " ' forms. Loss of 0.7 Trp in CCP(M1)-I and W5 1F-I, and 0.2 Trp in W 191F-I implies that R" is located on Trpl91 in CCP(M1)-I and W5 IF-I. Spontaneous decay of the F~"=o hemes back to ~e " ' , followed by reaction with 2 more equivalents of H202 after 24 h, resulted in a combined loss of 2.7 (CCP(MI)), 1.5 (W51F), and -1 (W191F) Trp. Also, addition of 6 equivalents of H202 to the resting ~e " ' enzymes resulted in loss of -2 Trps in CCP(M1) but only -1 in W51F and W 191F, suggesting that Trp51 becomes redox active in CCP(M1) when >2 equivalents of H202 are reduced. Addition of 20 equivalents of H202 resulted in a total loss of -4,2.5, and 2 Trp in CCP(MI), W51F, and W 191F, respectively. Activity loss largely paralleled Trp loss, and the residual activity of CCP(M1) and W5 lFexposed to 20 equivalents of H202 was 5-19%, while W191F exhibited -50% activity. SDS PAGE analysis revealed that oxidized CCP(M1) and W191F were 60-70% monomeric, and W51F 27% monomeric following its reaction with >2 equivalents H202. 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.

LC-MS/MS suggests that hole hopping in cytochrome c peroxidase protects its heme from oxidative modification by excess H2O2

“…Building on previous studies 16–23,26,32–35 that pinpointed a limited number of donors to the heme in the endogenous oxidation of Ccp1 by H 2 O 2 , here we identify an unprecedented number of donor residues (24) by characterizing their stable end products by in-depth LC-MS/MS analysis. This large number of proximal and distal donors delineates numerous hole-hopping pathways emanating from Ccp1's heme that protect it from irreversible modification.…”

Redesign of Cytochrome c Peroxidase into a Manganese Peroxidase: Role of Tryptophans in Peroxidase Activity

“…Oxidation of WTCcP residues during its reaction with hydrogen peroxide has been reported. Erman observed CcP dimerization through oxidized tyrosine residues (34), and English reported the loss of tryptophan and tyrosine residues as a result of the treatment of CcP with excess hydrogen peroxide (35,37). By replacing Phe with Trp in horseradish peroxidase at the corresponding position of Trp191 in CcP, Morimoto et al observed spectral features corresponding a ferryl and a Trp radical (46).…”

“…For CcP(MI, W191F) and CcP(MI, W51F, W191F), the transiently formed compound I decays to a species containing a ferryl oxo and protein radical. The location(s) of this radical in CcP mutants containing the Trp191Phe mutation has not been unambiguously identified (34)(35)(36)(37)52). Although Trp51 has been ruled out as the primary radical site in WTCcP, its close position to the heme makes it an attractive site for the radical in the W191F mutants.…”

“…However, in comparison to other peroxidases such as MnP, the C c P(MI, W191F) compound I lifetime ( t 1/2 < 14 ms) is extremely short when no substrate is present. Compound I was reduced by an endogenous donor to a ferryl species and unknown amino acid radical ( − ). While Trp51 is not the primary location of the radical in WTC c P containing Trp191, its close proximity to the heme makes it a potential alternate site in C c P mutants containing the Trp191Phe mutation.…”

Phenotypic analysis of the ccp1Δ and ccp1Δ-ccp1W191F mutant strains of Saccharomyces cerevisiae indicates that cytochrome c peroxidase functions in oxidative-stress signaling

“…Considering that CCP and CCP W191F exhibit very different properties in vitro [12,14,[19][20][21], phenotypic comparison of ccp1D and ccp1D-ccp1 W191F yeast is of much interest. Here we compare the growth and survival of unstressed and H 2 O 2 -challenged ccp1D, ccp1D-ccp1 W191F and wild-type S. cerevisiae cells in the W303-1B genetic background.…”