Iron is a fundamental element for virtually all forms of life. Despite its abundance, its bioavailability is limited, and thus, microbes developed siderophores, small molecules, which are synthesized inside the cell and then released outside for iron scavenging. Once inside the cell, iron removal does not occur spontaneously, instead this process is mediated by siderophore-interacting proteins (SIP) and/or by ferric-siderophore reductases (FSR). In the past two decades, representatives of the SIP subfamily have been structurally and biochemically characterized; however, the same was not achieved for the FSR subfamily. Here, we initiate the structural and functional characterization of FhuF, the first and only FSR ever isolated. FhuF is a globular monomeric protein mainly composed by α-helices sheltering internal cavities in a fold resembling the “palm” domain found in siderophore biosynthetic enzymes. Paramagnetic NMR spectroscopy revealed that the core of the cluster has electronic properties in line with those of previously characterized 2Fe–2S ferredoxins and differences appear to be confined to the coordination of Fe(III) in the reduced protein. In particular, the two cysteines coordinating this iron appear to have substantially different bond strengths. In similarity with the proteins from the SIP subfamily, FhuF binds both the iron-loaded and the apo forms of ferrichrome in the micromolar range and cyclic voltammetry reveals the presence of redox-Bohr effect, which broadens the range of ferric-siderophore substrates that can be thermodynamically accessible for reduction. This study suggests that despite the structural differences between FSR and SIP proteins, mechanistic similarities exist between the two classes of proteins.

Graphic abstract

中文翻译：

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召唤幽灵：FhuF 的结构和功能表征，一种来自大肠杆菌的铁载体还原酶

铁是几乎所有生命形式的基本元素。尽管其丰富，但其生物利用度是有限的，因此，微生物产生了铁载体，即小分子，它们在细胞内合成，然后释放到外面以清除铁。一旦进入细胞，铁去除不会自发发生，而是由铁载体相互作用蛋白 (SIP) 和/或铁-铁载体还原酶 (FSR) 介导。在过去的 20 年里，SIP 亚科的代表已经在结构和生物化学上进行了表征；然而，FSR 亚科没有达到同样的效果。在这里，我们启动了 FhuF 的结构和功能表征，这是有史以来第一个也是唯一一个分离出的 FSR。FhuF 是一种球状单体蛋白质，主要由 α 螺旋组成，这些 α 螺旋在类似于铁载体生物合成酶中发现的“棕榈”结构域的折叠中保护内部空腔。顺磁 NMR 光谱显示，簇的核心具有与先前表征的 2Fe-2S 铁氧还蛋白的电子特性一致，差异似乎仅限于还原蛋白质中 Fe(III) 的配位。特别是，与该铁配位的两个半胱氨酸似乎具有显着不同的键强度。与 SIP 亚家族的蛋白质相似，FhuF 在微摩尔范围内结合载铁和 apo 形式的铁色素，循环伏安法显示存在氧化还原-玻尔效应，这拓宽了可在热力学上进行还原的三价铁-铁载体底物的范围。这项研究表明，尽管 FSR 和 SIP 蛋白质之间存在结构差异，但两类蛋白质之间存在机制相似性。

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