LC-MS/MS suggests that hole hopping in cytochrome c peroxidase protects its heme from oxidative modification by excess H 2 O 2

We recently reported that cytochrome c peroxidase (Ccp1) functions as a H O sensor protein when H O levels rise in respiring yeast. The availability of its reducing substrate, ferrocytochrome c (Cyc ), determines whether Ccp1 acts as a H O sensor or peroxidase. For H O to serve as a signal it must modify its receptor so we employed high-performance LC-MS/MS to investigate in detail the oxidation of Ccp1 by 1, 5 and 10 M eq. of H O in the absence of Cyc to prevent peroxidase activity. We observe strictly heme-mediated oxidation, implicating sequential cycles of binding and reduction of H O at Ccp1's heme. This results in the incorporation of ∼20 oxygen atoms predominantly at methionine and tryptophan residues. Extensive intramolecular dityrosine crosslinking involving neighboring residues was uncovered by LC-MS/MS sequencing of the crosslinked peptides. The proximal heme ligand, H175, is converted to oxo-histidine, which labilizes the heme but irreversible heme oxidation is avoided by hole hopping to the polypeptide until oxidation of the catalytic distal H52 in Ccp1 treated with 10 M eq. of H O shuts down heterolytic cleavage of H O at the heme. Mapping of the 24 oxidized residues in Ccp1 reveals that hole hopping from the heme is directed to three polypeptide zones rich in redox-active residues. This unprecedented analysis unveils the remarkable capacity of a polypeptide to direct hole hopping away from its active site, consistent with heme labilization being a key outcome of Ccp1-mediated H O signaling. LC-MS/MS identification of the oxidized residues also exposes the bias of electron paramagnetic resonance (EPR) detection toward transient radicals with low O reactivity.