FET3P (FERROXIDASE) AND FTR1P (PERMEASE) IN IRON UPTAKE

这项研究的长期目标是确定酵母蛋白Fet3p对这种有机体高亲和力铁摄取的重要性的潜在机制。我们已发表的和初步的数据表明，Fet3p是人类铜蓝蛋白Hcp的结构和功能同系物，特别是Fet3p和Hcp共同催化的氧对Fe(II)的氧化，这两种蛋白在多铜氧化酶家族中唯一具有的铁氧合酶活性，是这两种蛋白为生物体铁稳态提供的基本功能。Fe3p是一种多核铜氧化酶，即它具有一个类型1或蓝色的铜(II)，以及由一个类型2的铜(II)和桥联的、双核的、反铁磁耦合的铜(II)对组成的三核铜簇合物。这些新数据有力地支持了我们的基本假设，即在Fet3p上的铁氧合酶位点产生的Fe(III)被输送到与HCP上的位点相当的保持位点，从那里该HCP Fe(III)被输送到人血浆中的转铁蛋白。在其在酿酒酵母铁吸收中的重要作用中，我们认为Fet3p将铁(III)传递到酵母中的铁渗透酶Ftr1蛋白，然后Ftr1蛋白将金属输送到细胞内。为了验证这一假说，我们提出了三个特定的目标：i)Fet3蛋白的结构和功能，其中我们将充分表征三个铜(II)位点的物理和氧化还原性质，以及它们在Fet3p和HCP唯一催化的亚铁氧基酶反应中的作用。Ii)Ftr1蛋白的结构和功能，其中我们将识别和确定Ftr1p中的残基对Fet3p产生的Fe(III)进入细胞所必需的特定作用。Iii)Fet3p和Ftr1p之间的铁通道，我们将测试这种Fe(III)从Aim I中确定的Fet3p上的结合位置转移到Aim II中确定的Ftr1p上的受体位置的可能机制。最近的数据表明，HCP对铁的稳态是必不可少的，特别是在神经组织中。Fet3p对于酵母中正常的铁代谢同样是必不可少的。在这两种情况下，它们的作用都被认为是将Fe(III)引导到Fe(III)结合蛋白，抑制Fe(II)的潜在氧化还原活性，同时保护Fe(III)不被水解。我们建议直接测试Fet3p的这个模型，并争辩说，我们的结果将与我们对HCP在人类铁稳态中的确切作用的理解高度相关，此外还提供了对酵母金属代谢至关重要的蛋白质的详细视图。

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.