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

We recently reported that cytochrome c peroxidase (Ccp1) functions as a H₂O₂ sensor protein when H₂O₂ levels rise in respiring yeast. The availability of its reducing substrate, ferrocytochrome c (Cyc^II), determines whether Ccp1 acts as a H₂O₂ sensor or peroxidase. For H₂O₂ 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 H₂O₂ in the absence of Cyc^II to prevent peroxidase activity. We observe strictly heme-mediated oxidation, implicating sequential cycles of binding and reduction of H₂O₂ 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 H₂O₂ shuts down heterolytic cleavage of H₂O₂ 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 H₂O₂ signaling. LC-MS/MS identification of the oxidized residues also exposes the bias of electron paramagnetic resonance (EPR) detection toward transient radicals with low O₂ reactivity.