Biological chemistry of heme-mediated peroxide signalling and heme trafficking

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Heme in hemoglobin transports oxygen but when it associates with other proteins, it performs different functions. For example, we recently demonstrated that the heme in cytochrome c peroxidase (Ccp1) signals when levels of hydrogen peroxide (a reactive oxygen species) in yeast mitochondria become potentially harmful. This occurs as cells deplete the glucose in their medium and switch from fermentation to respiration, which produces hydrogen peroxide as a by-product. Serendipitously, we noticed that heme-free Ccp1 exits respiring mitochondria, which at the same time accelerate their decomposition of hydrogen peroxide with oxygen generation. This is indicative of catalase activity and we showed that the heme from Ccp1 is transferred to catalase, an enzyme that uses heme to rapidly decompose hydrogen peroxide. We also showed using mass spectrometry that hydrogen peroxide weakens heme binding to Ccp1 by oxidizing the protein but that the heme remains undamaged.Such peroxide-induced heme mobilization has not been reported before so we aim to find out in atomic detail how this occurs in Ccp1. We will prepare mutant forms of Ccp1 to test possible mechanisms as well as model the reaction using computational methods to identify species that might not be stable enough to be detected experimentally. In characterizing the oxidation of Ccp1, we noted that its heme is wired' to many surface residues. We think that these residues locate binding sites for antioxidants present in the cell that protect Ccp1 or for compounds that Ccp1 converts to bioactive metabolites. We will examine the roles of these wires in detail and we will test if specific wires can be turned off by mutating residues along them. Heme is made in mitochondria but how it is transported to the many heme-requiring proteins in the cell remains a mystery. Inspired by our discovery that Ccp1's heme is transferred to catalase, we used a proteomics approach to discover other proteins involved in heme trafficking to or from Ccp1. We identified several proteins of interest, and we will explore heme transfer between them using sophisticated microscopy techniques. A hallmark of our research program is our use of yeast as a living test tube to guide our experiments and to discover new chemistry. Importantly, yeast and human cells share common signalling pathways so we will create key knowledge on hydrogen peroxide signalling and heme trafficking in health and disease that will contribute to drug discovery.