Lc-Ms/Ms Proteoform Profiling Exposes Cytochrome C Peroxidase Self-Oxidation In Mitochondria And Functionally Important Hole Hopping From Its Heme

LC-MS/MS profiling reveals that the proteoforms of cytochrome c peroxidase (Ccpl) isolated from respiring yeast mitochondria are oxidized at numerous Met, Trp, and Tyr residues. In vitro oxidation of recombinant Ccpl by H2O2 in the absence of its reducing substrate, ferrocytochrome c, gives rise to similar proteoforms, indicating uncoupling of Ccp1 oxidation and reduction in mitochondria. The oxidative modifications found in the Ccpl proteoforms are consistent with radical transfer (hole hopping) from the heme along several chains of redox-active residues (Trp, Met, Tyr). These modifications delineate likely hole-hopping pathways to novel substrate-binding sites. Moreover, a decrease in recombinant Ccpl oxidation by H2O2 in vitro in the presence of glutathione supports a protective role for hole hopping to this antioxidant. Isolation and characterization of extramitochondrial Ccpl proteoforms reveals that hole hopping from the heme in these proteoforms results in selective oxidation of the proximal heme ligand (H175) and heme labilization. Previously, we demonstrated that this labilized heme is recruited for catalase maturation (Kathiresan, M.; Martins, D.; English, A. M. Respiration triggers heme transfer from cytochrome c peroxidase to catalase in yeast mitochondria. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 17468-17473; DOI: 10.1073/pnas.1409692111). Following heme release, apoCcpl exits mitochondria, yielding the extramitochondrial proteoforms that we characterize here. The targeting of Ccpl for selective H175 oxidation may be linked to the phosphorylation status of Y153 close to the heme since pY153 is abundant in certain proteoforms. In sum, when insufficient electrons from ferrocytochrome c are available to Ccpl in mitochondria, hole hopping from its heme expands its physiological functions. Specifically, we observe an unprecedented hole-hopping sequence for heme labilization and identify hole-hopping pathways from the heme to novel substrates and to glutathione at Ccpl's surface. Furthermore, our results underscore the power of proteoform profiling by LC-MS/MS in exploring the cellular roles of oxidoreductases.