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

本研究的长期目标是确定酵母蛋白Fet3p对该生物体高亲和力铁摄取的重要性的机制。 我们已发表的初步数据表明，Fet3p是人铜蓝蛋白，hCp的结构和功能的同源物，特别是，铁（II）的氧化由Fet3p和hCp催化的双氧，铁氧化酶活性，这两种蛋白质，在家庭的多铜氧化酶，独特的，是必不可少的功能，这两种蛋白质提供有机体铁稳态。 Fet3p是一种多核铜氧化酶，也就是说，它有一个1型或“蓝色”Cu（II），和一个由一个2型Cu（II）和一个桥接的双核反铁磁耦合Cu（II）对组成的三核铜簇。这些新的数据有力地支持了我们的基本假设，即在Fet3p上的铁氧化酶位点产生的Fe（III）被引导到与hCp上的一个位点相当的保持位点，从该位点hCp Fe（III）被传递到人血浆中的转铁蛋白。 在其铁的吸收在酿酒酵母中的重要作用，我们建议，Fet3p提供此Fe（III）的铁渗透酶在酵母中，Ftr1蛋白，然后通道的金属进入细胞。 为了验证这一假设，我们提出了三个具体目标：I）Fet 3蛋白的结构和功能，其中我们将充分表征三个Cu（II）位点的物理和氧化还原性质以及它们在Fet 3 p与hCp沿着独特催化的亚铁氧化酶反应中的作用。II）Ftr1蛋白的结构和功能，其中我们将鉴定和确定Ftr1p中残基的特定作用，这些残基对于Fet3p产生的Fe（III）进入细胞至关重要。III）Fet3p和Ftr1p之间的铁交换，其中我们将测试Fe（III）从Aim I中鉴定的Fet3p上的结合位点转移到Aim II中鉴定的Ftr1p中的受体位点的可能机制。 最近的数据表明，hCp是必不可少的铁稳态，特别是在神经组织。Fet3p对酵母中的正常铁代谢同样至关重要。在这两种情况下，每个角色都被认为是Fe（III）与Fe（III）结合蛋白的通道，抑制Fe（II）的潜在氧化还原活性，同时屏蔽Fe（III）免于水解。 我们建议直接测试这个模型的Fet3p，并认为我们的研究结果将是高度相关的，我们的理解hCp在人体铁稳态的确切作用，除了提供一个详细的蛋白质酵母金属代谢必不可少的视图。

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.