Abstract

The stress response to exogenous H2O2 has provided significant insight into thiol-based cytosolic H2O2 sensors such as OxyR in Escherichia coli and its eukaryotic counterpart Gpx3/Yap1 in Saccharomyce cerevisiae. However, sensing of endogenously generated H2O2 in mitochondria, the main sites of H2O2 production in respiring yeast, is poorly documented. We have recently demonstrated that cytochrome c peroxidase (Ccp1) acts as a mitochondrial H2O2 sensor to balance reactive oxygen species (ROS) levels by regulating catalase A (Cta1) activity and attenuating the H2O2 stress response during aging. Since Ccp1 is the first heme-based H2O2 sensor to be identified, its mechanism of action at the molecular level is of much interest. This thesis reports on how H2O2 signals through Ccp1. Activity assays reveal the accumulation of catalytically inactive and heme-free Ccp1 outside mitochondria in respiring cells, when catalase activity starts to increase. Importantly, the burst in H2O2 generation during the switch from fermentation to respiration triggers heme-mediated oxidation by H2O2 of the proximal Fe ligand, H175. Formation of oxo-H175 weakens the heme-ligand interaction and labilizes the heme group, which is transferred either directly or via unidentified intermediate(s) to apoCta1. The nascent Cta1 activity detoxifies H2O2, and apoCcp1 which has conformational lability, undergoes reverse translocation to the vacuole and nucleus. Mass spectrometric characterization of heme-mediated H2O2-induced chemical and post-translational modifications in recombinant Ccp1 and Ccp1 isolated from yeast cells, respectively, has allowed us to identify the residues oxidized as well as to evaluate radical-transfer pathways in Ccp1 in response to both exogenous and endogenous H2O2. Importantly, the mass spectrometry-based approach described here provides a way to fully evaluate the oxidation profile of a protein. Although Cta1 was not found as a binding partner of GST-Ccp1 in GST pull-down assays, the antioxidant proteins, manganese superoxide dismutase (Sod2), thioredoxin peroxidase (Tsa1), the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the putative heme transporter Pet9 and heat shock proteins 70 and 90 were reproducibly found as Ccp1 binding partners. Combined, the results presented provide insights into the mechanism of H2O2 signaling in cells involving heme-mediated redox changes in contrast to the redox chemistry of thiols.