The long term objective of this research is to determine the mechanism underlying the essentiality of the yeast protein, Fet3p, to high affinity iron uptake in this organism. Our published and preliminary data demonstrate that Fet3p is a structural and functional homologue of human ceruloplasmin, hCp and, specifically, that the oxidation of Fe(II) by dioxygen catalyzed by both Fet3p and hCp, the ferroxidase activity that these two proteins, in the family of multicopper oxidases, uniquely has, is the essential function that both proteins provide to organismal iron homeostasis. Fet3p is a multinuclear copper oxidase, that is, it has one type 1 or """"""""blue"""""""" Cu(II), and a trinuclear copper cluster comprised of one type 2 Cu(II) and a bridged, binuclear, antiferromagnetically coupled Cu(II) pair. These new data strongly support our fundamental hypothesis that the Fe(III) generated at a ferroxidase site on Fet3p is channeled to a holding site equivalent to a site on hCp from which this hCp Fe(III) is delivered to transferrin in human plasma. In its essential role in iron uptake in Saccharomyces cerevisiae, we propose that Fet3p delivers this Fe(III) to the iron permease in yeast, the Ftr1 protein, which then channels the metal into the cell. To test this hypothesis we propose three Specific Aims: I) Structure and Function in the Fet3 Protein, in which we will fully characterize the physical and redox properties of the three Cu(II) sites and their role in the ferroxidase reaction that Fet3p, along with hCp, uniquely catalyzes. II) Structure and Function in the Ftr1 Protein, in which we will identify and determine the specific roles for the residues in Ftr1p essential to the channeling of Fet3p-generated Fe(III) into the cell. III) Iron Channeling between Fet3p and Ftr1p in which we will test possible mechanisms for the transfer of this Fe(III) from its binding site on Fet3p identified in Aim I to its receptor site in Ftr1p identified in Aim II. Recent data have shown that hCp is essential to iron homeostasis particularly in neural tissues. Fet3p is similarly essential to normal iron metabolism in yeast. In both cases the role each plays is proposed to be the channeling of Fe(III) to an Fe(III) binding protein, suppressing the potential redox activity of Fe(II) while shielding the Fe(III) from hydrolysis. We propose to test this model for Fet3p directly and argue that our results will be highly relevant to our understanding of the precise role of hCp in human iron homeostasis in addition to providing a detailed view of a protein essential to yeast metal metabolism.
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