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LC-MS/MS Proteoform Profiling Exposes Cytochrome c Peroxidase Self-Oxidation in Mitochondria and Functionally Important Hole Hopping from Its Heme

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journal contribution
posted on 27.08.2018 by Meena Kathiresan, Ann M. English
LC-MS/MS profiling reveals that the proteo­forms of cytochrome c peroxidase (Ccp1) isolated from respiring yeast mito­chondria are oxidized at numerous Met, Trp, and Tyr residues. In vitro oxidation of recombinant Ccp1 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 Ccp1 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 Ccp1 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 Ccp1 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, apoCcp1 exits mito­chondria, yielding the extra­mito­chondrial proteo­forms that we characterize here. The targeting of Ccp1 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 Ccp1 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 Ccp1’s surface. Furthermore, our results underscore the power of proteo­form profiling by LC-MS/MS in exploring the cellular roles of oxidoreductases.