Abstract

Independently of its peroxidase activity, yeast cytochrome c peroxidase (Ccp1) functions in H2O2 sensing and signaling, including regulation of heme catalase A, Cta1. Since the maturation of Cta1, a highly efficient H2O2 detoxifying enzyme, involves the recruitment of Ccp1’s heme in respiring yeast mitochondria, I aimed to track the heme status of Ccp1 in live cells spatiotemporally. To do this, I had to develop a tool to investigate when and where an endogenous heme-binding protein such as Ccp1 is heme-loaded or heme-free in live cells. As described in Chapter 2, analysis of GFP fluorescence lifetimes by time-correlated single photon counting (TCSPC) indicates that heme efficiently quenches green fluorescent protein (GFP) in a recombinant Ccp1-GFP fusion protein in vitro and the amplitude of the quenched lifetime increases linearly with heme binding to Ccp1. Thus, in Chapter 3, I used fluorescence lifetime imaging microscopy (FLIM) of cells chromosomally expressing the same Ccp1-GFP fusion under the control of Ccp1’s native promotor to show that Ccp1-GFP is fully heme-loaded and resides in the mitochondria of two-day respiring cells but more than half of Ccp1-GFP is heme-free and extra-mitochondrial in seven-day respiring cells. Since Ccp1 is the first heme-based H2O2-sensing protein to be identified and its W191F variant behaves as an amplified H2O2 sensor, the second goal of my research was to compare the H2O2-responsive proteins regulated by Ccp1 and Ccp1W191F in cells. Using mass spectrometry-based label-free proteomics, a switch of cellular activity from biosynthesis to defense and a redirection of carbohydrate metabolism to NADPH and trehalose production were found in both yeast strains on H2O2 challenge as reported in Chapter 4. However, cells expressing Ccp1W191F mounted a significantly more sustained response to H2O2 and the in vitro experiments described in Chapter 5 suggest that Ccp1W191F is a dedicated H2O2 sensing/signaling protein in cells. Overall, my research findings confirm that Ccp1 functions as a heme-donor protein in cells and that it acts as a regulator of the H2O2 stimulon.