Modification of cytochrome c peroxidase with hydrogen peroxide : characterization of the endogenous decay products at pH 8.0

The endogenous decay products of compound I at pH 8.0 were studied. Analysis included activity towards the model substrate, ferrocyanide, hydrogen peroxide titer, amino acid analysis and peptide mapping. The composite decay products showed a loss of activity towards ferrocyanide which coincides with the loss of the hydrogen peroxide titer. The optical spectrum showed loss of absorbtivity and a shift to longer wavelength as the hydrogen peroxide modification ratio was increased. SDS-PAGE showed that the products were not homogenous. Amino acid analysis indicated the loss of tryptophan and tyrosine residues. Concentration-dependent studies of the decay showed that there was a change in the activity of the decay products as the concentration of the enzyme was increased. Gel filtration studies at different enzyme concentrations showed changes in the distribution of products. These observations indicate the presence of bimolecular interactions during the decay process. Separation and characterization of the decay products produced at low concentration revealed two major monomeric components along with dimer and trimer species. All of the species showed reduced activity towards ferrocyanide and the loss of the hydrogen peroxide titer. The optical spectrum of the products showed perturbations in the heme environment. Amino acid analysis showed loss of tryptophan and tyrosine residues. The peptide map of the separated products showed modification of peptides which surround the methionine cluster at positions 230 and 231. These peptides include T25, which contains tryptophan 223, T26, which contains tyrosines 229 and 236, and T23, which contains tryptophan 191. Some of the more highly oxidized species showed loss of T7 which contains tryptophan 51. The dimer and trimer species show a loss of peak 28 in the peptide map. This peak contains tyrosine 42 which may be involved in the covalent linkage producing polymeric forms.