Defects in heme biosynthesis and late stages of erythropoiesis and erythroid cell maturation can lead to microcytic anemias, iron homeostasis malfunction with iron accumulation in the heart (e.g., hemochromatosis), and consequently to heart diseases. Ferrochelatase (FC) catalyzes the terminal step of heme biosynthesis, the insertion of ferrous iron (Fe2+) into protoporphyrin IX. While it is recognized that a mechanism must exit to prevent Fe2+ oxidation and directly deliver Fe2+ to FC, the jury is still out regarding the Fe2+ chaperone or transporter. Frataxin, a mitochondrial matrix Fe2+ chaperone, has emerged as a serious contender. Our previous studies led us to the identification of a docking site for a potential mitochondrial iron chaperone in the region of the catalytic Fe2+-porphryin insertion site of FC. Clearly, Fe2+ delivery to FC has to be coordinated with the binding of the porphyrin substrate for heme synthesis to occur. We propose to investigate whether these processes are orchestrated through specifically modulated protein interactions. The general hypothesis is: Frataxin, by maintaining iron as Fe2+, makes the physiologically relevant metal ion substrate readily available to FC via a mechanism that prevents alternative metal ions from being incorporated into the porphyrin ring. To test this hypothesis, we will address the following Specific Aims: 1.Define the interaction and binding kinetics between FC and frataxin. 2.Determine whether protoporphyrin IX modulates the interaction and how this interaction controls Fe2+ delivery and the FC reaction mechanism. A combination of experimental approaches, ranging from construction of FC and frataxin variants, transient kinetics, crosslinking and mass spectrometry, isothermal titration calorimetry, surface plasmon resonance to molecular docking, will be utilized to provide interpretations at a molecular level for the role of the FC-frataxin interaction in Fe2+ delivery and heme biosynthesis. The findings from the proposed studies will also establish whether the frataxin-FC interaction can be considered as a potential target for development of therapies for disorders associated with late stages of erythroid maturation.
|Effective start/end date||07/01/2013 → 06/30/2015|