Different pathways of radical translocation in yeast cytochrome c peroxidase and its W191F mutant on reaction with H₂O₂ suggest an antioxidant role

Abstract.

In the absence of exogenous donors, turnover of 10 molar equivalents of H₂O₂ by wild-type recombinant cytochrome c peroxidase [CCP(MI)] and its W191F mutant at pH 7.0 occurs by oxidation of endogenous donors on the polypeptide. No O₂ evolution was observed with either enzyme on reaction with 10 molar equivalents of H₂O₂, eliminating catalase-like activity ${\rm{(2H}}_{\rm{2}} {\rm{O}}_{\rm{2}} \buildrel {} \over \rightleftharpoons {\rm{O}}_{\rm{2}} + {\rm{2H}}_{\rm{2}} {\rm{O)}}$ , but O₂ evolution was observed when 100 molar equivalents of H₂O₂ were added to the enzymes. Protein dimers were observed by SDS-PAGE following H₂O₂ turnover by the peroxidases, and dimeric forms of CCP(MI) and CCP(W191) were isolated by gel-permeation chromatography. LC-ESI-MS analysis of the tryptic digests of the dimers revealed the previously reported T₆-T₆ crosslink and a new crosslink between T₆-T₂₆, but no T₂₆-T₂₆ crosslink. The crosslinked tryptic peptides contain the exposed tyrosine residues Tyr36, Tyr39 and Tyr42 (T₆), and Tyr229 and Tyr236 (T₂₆). Addition of a spin trap, 2-methyl-2-nitrosopropane (MNP), to the CCP(MI)/H₂O₂ reaction resulted in MNP labeling of peptides T₆, T₂₁ (which contains Tyr153) and T₂₆. MNP labeling of Tyr236 was found by sequencing peptide T₂₆. MNP labeling did not compete with dimerization of H₂O₂-oxidized CCP(W191F), suggesting that dityrosine formation in this mutant is very rapid owing to the high reactivity of radicals formed on T₆. H₂O₂-dependent formation of CCP-cytochrome c heterodimers was observed for both CCP(MI) and W191F in the presence of ferricytochrome c, the oxidized form of CCP's donor substrate. Interestingly, no H₂O₂-dependent cytochrome crosslinking to the W51F mutant was observed, even though this mutant underwent extensive homocrosslinking. The translocation of oxidizing equivalents from the heme to the surface residues of CCP is discussed in terms of an antioxidant role for CCP.