Elucidating the Biosynthesis and Transport of Heme A

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DESCRIPTION (provided by applicant): Cytochrome c oxidase is the terminal oxidase in all plants, animals, aerobic yeasts, and some bacteria. Catalyzing the reduction of molecular oxygen to water concomitant with the pumping of protons across the inner mitochondrial membrane, cytochrome c oxidase generates a proton gradient responsible for approximately 50 percent of the ATP formed during eukaryotic aerobic metabolism. Heme A is an obligatory cofactor in eukaryotic cytochrome c oxidase, present at both an electron transfer site and the site of oxygen reduction. Heme A differs from heme B (protoheme) in that a farnesyl moiety has been added to one of the vinyl groups, and a methyl substituent has been oxidized to an aldehyde. These conversions (both of which are required to obtain active cytochrome c oxidase) are catalyzed by heme 0 synthase and heme A synthase, respectively. Surprisingly, despite the obvious importance of heme A to both cytochrome c oxidase and the energy transduction pathway, very little is known about how heme A is synthesized, how it is transported, or how it is inserted into cytochrome c oxidase.The focus of this research is to elucidate the biosynthesis of heme A and its method of transport, including the formation of associated multi-component complexes. Preliminary data obtained in this lab demonstrates that heme A synthase alters the activity of heme 0 synthase, suggesting that the two enzymes are part of a larger protein complex. We have also obtained the first direct evidence indicating that heme A synthase represents a novel heme-containing oxygenase. To confirm these tantalizing results and to enhance our understanding of heme A biosynthesis and transport, an interdisciplinary research plan has been developed. Biochemical and molecular approaches will be used to define specific macromolecular complexes and protein-protein interactions involving heme A synthase. The proteins hypothesized to interact with heme A synthase are 1) heme 0 synthase, 2) two COX1 translation activators of the mitochondrial ribosome complex, and 3) subunit I of cytochrome c oxidase. In addition, a variety of chemical and spectroscopic techniques will be employed to 1) fully characterize the active site of heme A synthase, 2) determine the nature of the active oxidant, and 3) elucidate the mechanism of heme A biosynthesis. Combined, the proposed experiments represent a comprehensive attack on heme A synthase, thus providing fundamental information about the assembly of cytochrome c oxidase and further insight into biological 02 activation.