The overall goals of the proposed work are to obtain data that will allow descriptions at the molecular level how the terminal two heme synthetic pathway enzymes, protoporphyrinogen oxidase (PRO) and ferrochelatase, function. The goals of the current proposal are to define catalytic and structural features of human PRO and ferrochelatase and bacterial oxygen-independent PRO. Potential protein-protein interactions involving these enzymes as well as cytochrome c and frataxin will be examined. The data obtained will describe the catalytic consequences of naturally occurring mutations in humans that lead to the inherited diseases known as porphyrias, and this should be of value in patient treatment and counseling. The data will also identify and characterize any key differences that exist between the human and microbial enzymes. These results may set the stage for development of new classes of antimicrobial agents.
Specific aims of this proposal are to: 1.) define structure-function characteristics of human PRO through the use of kinetics, site-directed mutagenesis, and structure-based studies, determine if cytochrome c is a bona fide electron acceptor for PPO and characterize this interaction, and isolate and characterize bacterial oxygen-independent protoporphyrinogen oxidase. 2.) define structure-function characteristics of ferrochelatase through the use of kinetics, site-directed mutagenesis, and structure-based studies, characterize the ferrochelatase:frataxin interaction and determine its relevance to ERR patients with ringed sideroblasts, and characterize the [2Fe-2S] cluster in animal and bacterial ferrochelatases through site-directed mutagenesis and physical examination. 3.) identify and characterize protein-protein interactions between ferrochelatase and PPO.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Erythrocyte and Leukocyte Biology Study Section (ELB)
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Bishop, Terry Rogers
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University of Georgia
Schools of Arts and Sciences
United States
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Dailey, Harry A; Meissner, Peter N (2013) Erythroid heme biosynthesis and its disorders. Cold Spring Harb Perspect Med 3:a011676
Medlock, Amy E; Najahi-Missaoui, Wided; Ross, Teresa A et al. (2012) Identification and characterization of solvent-filled channels in human ferrochelatase. Biochemistry 51:5422-33
Hamza, Iqbal; Dailey, Harry A (2012) One ring to rule them all: trafficking of heme and heme synthesis intermediates in the metazoans. Biochim Biophys Acta 1823:1617-32
Chen, Caiyong; Samuel, Tamika K; Sinclair, Jason et al. (2011) An intercellular heme-trafficking protein delivers maternal heme to the embryo during development in C. elegans. Cell 145:720-31
Dailey, Harry A; Septer, Alecia N; Daugherty, Lauren et al. (2011) The Escherichia coli protein YfeX functions as a porphyrinogen oxidase, not a heme dechelatase. MBio 2:e00248-11
Boynton, Tye O; Gerdes, Svetlana; Craven, Sarah H et al. (2011) Discovery of a gene involved in a third bacterial protoporphyrinogen oxidase activity through comparative genomic analysis and functional complementation. Appl Environ Microbiol 77:4795-801
Dailey, Tamara A; Boynton, Tye O; Albetel, Angela-Nadia et al. (2010) Discovery and Characterization of HemQ: an essential heme biosynthetic pathway component. J Biol Chem 285:25978-86
Chen, Wen; Dailey, Harry A; Paw, Barry H (2010) Ferrochelatase forms an oligomeric complex with mitoferrin-1 and Abcb10 for erythroid heme biosynthesis. Blood 116:628-30
Boynton, Tye O; Daugherty, Lauren E; Dailey, Tamara A et al. (2009) Identification of Escherichia coli HemG as a novel, menadione-dependent flavodoxin with protoporphyrinogen oxidase activity. Biochemistry 48:6705-11
Shepherd, M; Dailey, H A (2009) Peroxidase activity of cytochrome C facilitates the protoporphyrinogen oxidase reaction. Cell Mol Biol (Noisy-le-grand) 55:6-14

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