Liu Fange, Geng Jiafeng, Gumpper Ryan H, Barman Arghya, Davis Ian, Ozarowski Andrew, Hamelberg Donald, Liu Aimin
Department of Chemistry, Georgia State University, Atlanta, Georgia 30303; Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, Georgia 30303.
Department of Chemistry, Georgia State University, Atlanta, Georgia 30303; Molecular Basis of Disease Program, Georgia State University, Atlanta, Georgia 30303.
J Biol Chem. 2015 Jun 19;290(25):15621-15634. doi: 10.1074/jbc.M115.650259. Epub 2015 Apr 27.
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.
红素氧还蛋白基序存在于超过74000个蛋白质序列和2000个结构中，但已知功能的却很少。在来自单细胞而非多细胞来源的3-羟基邻氨基苯甲酸3,4-双加氧酶（HAO）中，一个二级的、非催化性的、类似红素氧还蛋白的铁位点是保守的。通过在细菌HAO的两个金属结合位点中填充各种金属，利用动力学、光谱学、晶体学和计算方法研究了类似红素氧还蛋白位点的结构与功能关系。结果表明，优先存在的第一种金属与催化位点结合，而非类似红素氧还蛋白的位点，当催化位点被占据时，该位点选择性地结合铁。此外，结合在类似红素氧还蛋白位点上的铁离子可通过分子间转移机制轻松传递至无金属HAO的空催化位点。通过金属分析和催化活性测量，我们表明下游代谢中间体可选择性地去除催化铁。由于原核HAO通常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代谢中间体的途径中，当催化过程中催化中心丢失铁时，类似红素氧还蛋白的位点可能是催化中心的辅助铁源。基于这项生化研究提出了一个备用轮胎概念，该概念为铁依赖酶中的铁硫中心开辟了一个潜在的新功能范式，即作为瞬时铁结合和穿梭位点以确保催化位点的完全金属负载。
