FERROCHELATASE AND THE TERMINAL STEP HEME BIOSYNTHESIS

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THIS IS A SHANNON AWARD PROVIDING PARTIAL SUPPORT FOR THE RESEARCH PROJECTS THAT FALL SHORT OF THE ASSIGNED INSTITUTE'S FUNDING RANGE BUT ARE IN THE MARGIN OF EXCELLENCE. THE SHANNON AWARD IS INTENDED TO PROVIDE SUPPORT TO TEST THE FEASIBILITY OF THE APPROACH; DEVELOP FURTHER TESTS AND REFINE RESEARCH TECHNIQUES; PERFORM SECONDARY ANALYSIS OR AVAILABLE DATA SETS; OR CONDUCT DISCRETE PROJECTS THAT CAN DEMONSTRATE THE PI'S RESEARCH CAPABILITIES OR LEND ADDITIONAL WEIGHT TO AN ALREADY MERITORIOUS APPLICATION. THE ABSTRACT BELOW IS TAKEN FROM THE ORIGINAL DOCUMENT SUBMITTED BY THE PRINCIPAL INVESTIGATOR. DESCRIPTION: The long-term objectives of this program are to understand the structure, function and catalytic mechanism of ferrochelatase, the terminal enzyme of the heme biosynthetic pathway, and their relationship to the regulation of heme biosynthesis in differentiating erythrocytes. Understanding the structure, function and regulation of ferrochelatase, at the gene and protein levels, is essential both for basic studies of erythroid differentiation and for elucidation of the pathophysiology of genetic diseases, such as erythropoietic protoporphyria. Further, the understanding, at the molecular level, of how enzymes of the heme biosynthetic pathway function and are regulated will provide the molecular basis of the porphyrin overproduction associated with hepatocellular carcinomas. Three recent major developments and findings in the principal investigator's laboratory, namely, 1) the development of a new overexpression system for ferrochelatase; 2) the identification of a [2Fe-2S] cluster in mammalian ferrochelatase; and 3) the identification that ferrochelatase exhibits IRE-binding and can repress the translation of the mRNA for the erythroid 5-aminolevulinate synthase, the first enzyme of the heme biosynthetic pathway, have opened new and challenging avenues for ferrochelatase and heme biosynthesis research. Projected studies will utilize chemical, biochemical, physical and molecular biological approaches to address the following specific aims: 1) To characterize the [2Fe-2S] cluster, by defining its spectroscopic properties and identifying the ferrochelatase amino acid residues essential for its binding; 2) To evaluate the possible role of the [2F2-2S] cluster in ferrochelatase activity; 3) To assess the role of RNA (i.e., IRE)-ferrochelatase interactions in the regulation of heme biosynthesis in differentiating erythrocytes; and 4) To recharacterize kinetically the recombinant murine ferrochelatase, under strictly anaerobic conditions and to identify the amino acids essential for binding of the substrates, using site-directed mutagenesis in combination with spectroscopic investigations.