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

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 (Fe^IV double bond O,R⁺) formed on addition of 2 equivalents of H₂O₂, exhibited decreased fluorescence relative to the Fe^III 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 Fe^IV double bond O hemes back to Fe^III, 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 Fe^III 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.