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对这种生物高亲和力铁摄取的重要性的机制。我们发表的和初步的数据表明，Fet3p是人类铜蓝蛋白hCp的结构和功能同源物，特别是，fe3p和hCp都催化了铁（II）的双氧氧化，这两种蛋白在多铜氧化酶家族中具有独特的氧化铁酶活性，是这两种蛋白提供机体铁稳态的基本功能。Fet3p是一种多核铜氧化酶，也就是说，它有一个1型或“蓝色”铜（II），以及一个由2型铜（II）和一个桥接、双核、反铁磁偶联的铜（II）对组成的三核铜簇。这些新数据有力地支持了我们的基本假设，即在Fet3p上的铁氧化酶位点产生的铁（III）被输送到一个与hCp上的一个位点相当的保持位点，hCp上的铁（III）从这个位点被输送到人血浆中的转铁蛋白中。在酿酒酵母中，Fet3p在铁摄取中起着重要作用，我们提出，Fet3p将铁（III）传递给酵母中的铁渗透酶，即Ftr1蛋白，然后将金属导入细胞。为了验证这一假设，我们提出了三个特定的目标：1)fe3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特性及其在fe3p和hCp独特催化的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，这些残基对于将fet3p生成的Fe（III）输送到细胞中至关重要。III) fe3p和Ftr1p之间的铁通道，我们将测试铁（III）从Aim I中鉴定的fe3p结合位点转移到Aim II中鉴定的Ftr1p受体位点的可能机制。最近的数据表明，hCp对铁稳态至关重要，特别是在神经组织中。Fet3p对酵母正常的铁代谢同样重要。在这两种情况下，每种作用都被认为是铁（III）到铁（III）结合蛋白的通道，抑制铁（II）的潜在氧化还原活性，同时屏蔽铁（III）免于水解。我们建议直接对Fet3p模型进行测试，并认为我们的结果将与我们对hCp在人体铁稳态中的确切作用的理解高度相关，此外还提供了酵母金属代谢必需蛋白质的详细视图。

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.