An Iron Reservoir to the Catalytic Metal: THE RUBREDOXIN IRON IN AN EXTRADIOL DIOXYGENASE

作者：Liu Fange; Geng Jiafeng; Gumpper Ryan H; Barman Arghya; Davis Ian; Ozarowski Andrew; Hamelberg Donald; Liu Aimin^*

来源：JOURNAL OF BIOLOGICAL CHEMISTRY, 2015, 290(25): 15621-15634.

DOI：10.1074/jbc.M115.650259

• computation
• dioxygenase
• iron-sulfur protein
• metabolism
• tryptophan
• iron reservoir
• kynurenine

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.

• 出版日期2015-6-19