An Iron Reservoir to the Catalytic Metal: THE RUBREDOXIN IRON IN AN EXTRADIOL DIOXYGENASE
An Iron Reservoir to the Catalytic Metal: THE RUBREDOXIN IRON IN AN EXTRADIOL DIOXYGENASE
复制标题
DOI：
10.1074/jbc.m115.650259
复制
发表时间：
2015-06-19
影响因子：
4.8
通讯作者：
Liu, Aimin
中科院分区：
文献类型：
--
作者：
Liu, Fange;Geng, Jiafeng;Liu, Aimin
背景：在3-羟基苯甲酸3,4-双加氧酶中存在一个功能未知的辅助[Fe(Cys)(4)]中心。结果：观察到分子间铁从[Fe(Cys)(4)]位点到催化位点的穿梭。结论：当催化金属在代谢过程中被剥离时，红霉素样结构域是催化位点的铁储存库。意义：提出了铁-硫中心的铁穿梭机制。rubredoxin基序存在于超过74,000个蛋白质序列和2,000个结构中，但很少有已知的功能。在3-羟基苯甲酸3,4-双加氧酶（HAO）中，一个二级的、非催化的红霉素样铁位点是保守的，来自单细胞来源，而不是多细胞来源。通过细菌HAO中两个金属结合位点与各种金属的结合，采用动力学、光谱、晶体学和计算方法研究了红霉素样位点的结构和功能关系。结果表明，第一种金属优先结合催化位点，而不是红霉素样位点，当催化位点被占用时，红霉素样位点选择性地结合铁。此外，结合到红霉素样位点的铁离子很容易通过分子间转移机制传递到无金属HAO的空催化位点。通过使用金属分析和催化活性测量，我们表明下游代谢中间体可以选择性地去除催化铁。由于原核HAO对细胞存活至关重要，因此需要确保其活性。这些结果表明，在具有金属螯合特性的代谢中间体的催化过程中，当红霉素样位点在催化过程中丢失时，它可能是催化中心的辅助铁源。基于这一生化研究提出了备用轮胎的概念，这一概念为铁依赖性酶中的铁硫中心作为瞬态铁结合和穿梭位点以确保催化位点的全金属负载开辟了一个潜在的新功能范式。
Background: An accessory [Fe(Cys)(4)] center of unknown function is present in 3-hydroxyanthranilate 3,4-dioxygenase. Results: An intermolecular iron shuttling from the [Fe(Cys)(4)] site to the catalytic site is observed. Conclusion: The rubredoxin-like domain is an iron reservoir for the catalytic site when the catalytic metal becomes stripped during metabolic events. Significance: An iron shuttling mechanism is proposed for the iron-sulfur center.The rubredoxin motif is present in over 74,000 protein sequences and 2,000 structures, but few have known functions. A secondary, non-catalytic, rubredoxin-like iron site is conserved in 3-hydroxyanthranilate 3,4-dioxygenase (HAO), from single cellular sources but not multicellular sources. Through the population of the two metal binding sites with various metals in bacterial HAO, the structural and functional relationship of the rubredoxin-like site was investigated using kinetic, spectroscopic, crystallographic, and computational approaches. It is shown that the first metal presented preferentially binds to the catalytic site rather than the rubredoxin-like site, which selectively binds iron when the catalytic site is occupied. Furthermore, an iron ion bound to the rubredoxin-like site is readily delivered to an empty catalytic site of metal-free HAO via an intermolecular transfer mechanism. Through the use of metal analysis and catalytic activity measurements, we show that a downstream metabolic intermediate can selectively remove the catalytic iron. As the prokaryotic HAO is often crucial for cell survival, there is a need for ensuring its activity. These results suggest that the rubredoxin-like site is a possible auxiliary iron source to the catalytic center when it is lost during catalysis in a pathway with metabolic intermediates of metal-chelating properties. A spare tire concept is proposed based on this biochemical study, and this concept opens up a potentially new functional paradigm for iron-sulfur centers in iron-dependent enzymes as transient iron binding and shuttling sites to ensure full metal loading of the catalytic site.