Functionality of the three-site ferroxidase center of Escherichia coli bacterial ferritin (EcFtnA).
Functionality of the three-site ferroxidase center of Escherichia coli bacterial ferritin (EcFtnA).
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DOI：
10.1021/bi401517f
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发表时间：
2014-01-28
期刊：
影响因子：
2.9
通讯作者：
Chasteen, N. D.
中科院分区：
文献类型：
--
作者：
Bou-Abdallah, F.;Yang, H.;Awomolo, A.;Cooper, B.;Woodhall, M. R.;Andrews, S. C.;Chasteen, N. D.
在大肠杆菌中发现至少三种铁蛋白，含血红素的细菌铁蛋白（EcBFR）和两种非血红素的细菌铁蛋白（EcFtnA和EcFtnB）。除了二铁亚铁氧化酶中心的保守的A-和B-位点之外，EcFtnA在二铁位点附近具有未知功能的第三个铁结合位点（C-位点）。在目前的工作中，铁氧化和沉积在EcFtnA的复杂的化学已被进一步定义通过结合血氧，pH值统计，停止流动和传统的动力学，紫外可见，荧光和EPR光谱测量的野生型蛋白质和定点变体的A-，B-和C-网站。数据显示，虽然H2O2是EcFtnA中的分子氧还原的产物，并且氧化以Fe（II）/O2~3：1的化学计量发生，但产生的大部分H2O2在随后的反应中以2：1的Fe（II）/H2O2化学计量消耗，从而抑制羟基自由基的形成。虽然A-和B-位点对于快速铁氧化是必不可少的，但C-位点减慢氧化并抑制铁氧化酶中心的铁周转。一个酪氨酰基自由基，分配到Tyr24附近的铁氧化酶中心，铁氧化过程中形成的蛋白质的功能和其可能的意义进行了讨论。总的来说，数据表明，有多个铁的氧化途径在EcFtnA与O2和H2O2作为氧化剂。此外，我们的数据不支持一个普遍的机制，铁氧化在所有的铁蛋白，即C-网站作为过境网站，最近提出的。
At least three ferritins are found in the bacterium Escherichia coli, the heme-containing bacterioferritin (EcBFR) and two non-heme bacterial ferritins (EcFtnA and EcFtnB). In addition to the conserved A- and B-sites of the diiron ferroxidase center, EcFtnA has a third iron-binding site (the C-site) of unknown function that is nearby the diiron site. In the present work, the complex chemistry of iron oxidation and deposition in EcFtnA has been further defined through a combination of oximetry, pH stat, stopped-flow and conventional kinetics, UV-visible, fluorescence and EPR spectroscopic measurements on the wild-type protein and site-directed variants of the A-, B- and C-sites. The data reveal that, while H2O2 is a product of dioxygen reduction in EcFtnA and oxidation occurs with a stoichiometry of Fe(II)/O2 ~ 3:1, most of the H2O2 produced is consumed in subsequent reactions with a 2:1 Fe(II)/H2O2 stoichiometry, thus suppressing hydroxyl radical formation. While the A- and B-sites are essential for rapid iron oxidation, the C-site slows oxidation and suppresses iron turnover at the ferroxidase center. A tyrosyl radical, assigned to Tyr24 near the ferroxidase center, is formed during iron oxidation and its possible significance to the function of the protein is discussed. Taken as a whole, the data indicate that there are multiple iron-oxidation pathways in EcFtnA with O2 and H2O2 as oxidants. Furthermore, our data do not support a universal mechanism for iron oxidation in all ferritins whereby the C-site acts as transit site, as recently proposed.
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