Reaction paths of iron oxidation and hydrolysis in horse spleen and recombinant human ferritins.
Reaction paths of iron oxidation and hydrolysis in horse spleen and recombinant human ferritins.
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DOI：
10.1021/bi973128a
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发表时间：
1998-06
期刊：
影响因子：
2.9
通讯作者：
Xiaoke Yang;Yu Chen-Barrett;Paolo Arosio;N. Chasteen
中科院分区：
文献类型：
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作者：
Xiaoke Yang;Yu Chen-Barrett;Paolo Arosio;N. Chasteen
采用紫外-可见光谱法、电极血氧饱和度测定法和pH恒温法研究了马脾铁蛋白（HoSF）和重组人H链和L链铁蛋白（HuHF和HuLF）中Fe（II）的氧化和水解。测量了适当的测试反应和电极响应，建立了用于铁蛋白铁吸收动力学研究的氧电极/pH恒温器的可靠性。化学计量比，Fe（II）/O2和H+/Fe（II），和氧的吸收率和质子的生产率同时测量作为铁负载的蛋白质的函数。数据表明，二铁亚铁氧化酶网站和矿物表面催化氧化的Fe（II）之间有明显的区别。由铁氧化酶位点引起的氧化/水解反应首次被确定，并由2Fe 2 + + O2 + 3 H2O--> [Fe 2 O（OH）2]2+ + H2 O2 + 2 H+给出，其中[Fe 2 O（OH）2]2+代表水解的双核铁（III）中心，根据紫外光谱滴定数据和吸收带最大值，推测该中心是μ-氧桥物种。铁从铁氧化酶位置向矿物核心的转移现已确定为[Fe_2 O（OH）_2]~（2+）+H_2 O-> 2FeOOH（核心）+2 H ~+。对于HoSF和HuHF两者，观察到蛋白质铁氧化酶活性随时间的再生，这与它们具有酶性质一致，并且通过较高的pH促进（7.0）温度（37 ℃）和L-亚基的存在，并在10分钟内完成。与以前的研究雅阁，矿物表面反应为4Fe ~（2+）+O ~（2+）+6 H ~（2 O-）> 4FeOOH（核）+ 8H ~+。随着蛋白质逐渐获得铁，氧化/水解越来越多地从铁氧化酶位点转移到基于矿物表面的机制，减少H2 O2的产生。
UV-visible spectroscopy, electrode oximetry, and pH stat were used to study Fe(II) oxidation and hydrolysis in horse spleen ferritin (HoSF) and recombinant human H-chain and L-chain ferritins (HuHF and HuLF). Appropriate test reactions and electrode responses were measured, establishing the reliability of oxygen electrode/pH stat for kinetics studies of iron uptake by ferritin. Stoichiometric ratios, Fe(II)/O2 and H+/Fe(II), and rates of oxygen uptake and proton production were simultaneously measured as a function of iron loading of the protein. The data show a clear distinction between the diiron ferroxidase site and mineral surface catalyzed oxidation of Fe(II). The oxidation/hydrolysis reaction attributed to the ferroxidase site has been determined for the first time and is given by 2Fe2+ + O2 + 3H2O --> [Fe2O(OH)2]2+ + H2O2 + 2H+ where [Fe2O(OH)2]2+ represents the hydrolyzed dinuclear iron(III) center postulated to be a mu-oxo-bridged species from UV spectrometric titration data and absorption band maxima. The transfer of iron from the ferroxidase site to the mineral core has been now established to be [Fe2O(OH)2]2+ + H2O --> 2FeOOH(core) + 2H+. Regeneration of protein ferroxidase activity with time is observed for both HoSF and HuHF, consistent with their having enzymatic properties, and is facilitated by higher pH (7.0) and temperature (37 degreesC) and by the presence of L-subunit and is complete within 10 min. In accord with previous studies, the mineral surface reaction is given by 4Fe2+ + O2 + 6H2O --> 4FeOOH(core) + 8H+. As the protein progressively acquires iron, oxidation/hydrolysis increasingly shifts from a ferroxidase site to a mineral surface based mechanism, decreasing the production of H2O2.