Kwok E Y, Stoj C S, Severance S, Kosman D J
Department of Biochemistry, The University at Buffalo, 140 Farber Hall, Buffalo, NY 14214, USA.
J Inorg Biochem. 2006 May;100(5-6):1053-60. doi: 10.1016/j.jinorgbio.2005.11.015. Epub 2006 Jan 4.
High affinity iron uptake in fungi is supported by a plasma membrane protein complex that includes a multicopper ferroxidase enzyme and a ferric iron permease. In Saccharomyces cerevisiae, this complex is composed of the ferroxidase Fet3p and the permease Ftr1p. Fe(II) serves as substrate for Fe-uptake by being substrate for Fet3p; the resulting Fet3p-produced Fe(III) is then transported across the membrane via Ftr1p. A model of metabolite channeling of this Fe(III) is tested here by first constructing and kinetically characterizing in Fe-uptake two Fet3p-Ftr1p chimeras in which the multicopper oxidase/ferroxidase domain of Fet3p has been fused to the Ftr1p iron permease. Although the bifunctional chimeras are as kinetically efficient in Fe-uptake as is the wild type two-component system, they lack the adaptability and fidelity in Fe-uptake of the wild type. Specifically, Fe-uptake through the Fet3p, Ftr1p complex is insensitive to a potential Fe(III) trapping agent - citrate - whereas Fe-uptake via the chimeric proteins is competitively inhibited by this Fe(III) chelator. This inhibition does not appear to be due to scavenging Fet3p-produced Fe(III) that is in equilibrium with bulk solvent but could be due to leakiness to citrate found in the bifunctional but not the two-component system. The data are consistent with a channeling model of Fe-trafficking in the Fet3p, Ftr1p complex and suggest that in this system, Fet3p serves as a redox sieve that presents Fe(III) specifically for permeation through Ftr1p.
真菌中高亲和力铁摄取由一种质膜蛋白复合物支持，该复合物包括一种多铜铁氧化酶和一种高铁通透酶。在酿酒酵母中，这种复合物由铁氧化酶Fet3p和通透酶Ftr1p组成。Fe(II)作为Fet3p的底物用于铁摄取；然后Fet3p产生的Fe(III)通过Ftr1p跨膜运输。本文通过首先构建两种Fet3p-Ftr1p嵌合体并对其铁摄取进行动力学表征来测试这种Fe(III)代谢物通道模型，其中Fet3p的多铜氧化酶/铁氧化酶结构域已与Ftr1p铁通透酶融合。尽管双功能嵌合体在铁摄取方面与野生型双组分系统在动力学上一样高效，但它们在铁摄取方面缺乏野生型的适应性和保真度。具体而言，通过Fet3p、Ftr1p复合物的铁摄取对潜在的Fe(III)捕获剂柠檬酸盐不敏感，而通过嵌合蛋白的铁摄取受到这种Fe(III)螯合剂的竞争性抑制。这种抑制似乎不是由于清除与大量溶剂处于平衡状态的Fet3p产生的Fe(III)，而是可能由于双功能系统中存在但双组分系统中不存在的对柠檬酸盐的渗漏。这些数据与Fet3p、Ftr1p复合物中铁运输的通道模型一致，并表明在该系统中，Fet3p作为一种氧化还原筛，特异性地呈现Fe(III)以便通过Ftr1p渗透。
