Aerobic respiration is a key energy-producing pathway in many prokaryotes and virtually all eukaryotes. The final step of aerobic respiration is most commonly catalyzed by heme-copper oxidases embedded in the cytoplasmic or mitochondrial membrane. The majority of these terminal oxidases are aa3 cytochrome c oxidases, meaning that the heme cofactor is a modified heme known as heme a. Despite the critical role of heme a in aerobic respiration, many details of the catalytic mechanism by which heme b, the prototypical cellular heme, is converted to heme o (a biosynthetic intermediate) and then to heme a have yet to be elucidated. The mechanism of heme transfer among the enzymes in the heme a biosynthetic pathway also remains unclear. In this dissertation, I report on my investigation of the protein-protein interactions of heme a synthase and the catalytic mechanism of this enzyme. Chapter 1 provides an overview of the structural and biochemical data that is currently available for heme o synthase and heme a synthase and also summarizes what is currently known about prenylated heme trafficking. Chapter 2 reports on our investigations of the protein-protein interactions of heme a synthase in Saccharomyces cerevisiae, a model eukaryote. Surprisingly, we found that heme a synthase can interact with the cytochrome bc1 complex even in the absence of cytochrome c oxidase. Finally, Chapter 3 summarizes our study of the catalytic mechanism of heme a synthase using both prokaryotic and eukaryotic heme a synthase variants. Our data suggest that a conserved glutamate stabilizes a carbocation that is proposed to form during catalysis. Overall, this dissertation contributes to our understanding of how heme a synthase functions at the catalytic level and as a participant in intracellular heme trafficking.
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