Iron-sulfur cluster disassembly in the FNR protein of Escherichia coli by O-2: [4Fe-4S] to [2Fe-2S] conversion with loss of biological activity
Iron-sulfur cluster disassembly in the FNR protein of Escherichia coli by O-2: [4Fe-4S] to [2Fe-2S] conversion with loss of biological activity
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DOI：
10.1073/pnas.94.12.6087
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发表时间：
1997-06-10
影响因子：
11.1
通讯作者：
Kiley, PJ
中科院分区：
文献类型：
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作者：
Khoroshilova, N;Popescu, C;Kiley, PJ
转录因子FNR（富马酸硝酸盐还原）在大肠杆菌中氧气稀缺时作为一种全局转录调节因子发挥作用需要铁硫（Fe - S）簇的存在。为了确定FNR活性形式中存在的Fe - S簇的氧化态和类型，我们用穆斯堡尔光谱学研究了厌氧纯化的FNR。我们的数据显示，这种形式的FNR含有一个[4Fe - 4S]²⁺簇（δ = 0.45 mm/s；ΔE_Q = 1.22 mm/s），并且当FNR暴露于空气中时，[4Fe - 4S]²⁺簇会迅速被破坏。在这些条件下，黄绿色的FNR活性形式变成深红色；对硫化物的分析表明，70%的不稳定硫化物仍然存在，这表明Fe - S簇已转化为一种不同的形式。然而，通过电子顺磁共振（EPR）几乎没有检测到[3Fe - 4S]簇。根据穆斯堡尔光谱学，在暴露于空气17分钟后，[4Fe - 4S]²⁺簇以约60%的产率转化为一个[2Fe - 2S]²⁺簇（δ = 0.28 mm/s；ΔE_Q = 0.58 mm/s）。FNR的[2Fe - 2S]²⁺簇形式对氧气稳定得多，但无法维持生物活性（例如，DNA结合）。然而，当加入半胱氨酸（Cys）、铁（Fe）、二硫苏糖醇（DTT）和NifS蛋白时，[2Fe - 2S]²⁺形式的DNA结合能力以及[4Fe - 4S]²⁺簇的特征吸收光谱可以在很大程度上恢复。含有[2Fe - 2S]²⁺簇的FNR形式是否具有任何生物学意义，例如作为一种在体内比脱辅基蛋白更迅速转化为活性形式的中间产物，还有待确定。
The transcription factor FNR (fumarate nitrate reduction) requires the presence of an iron-sulfur (Fe-S) cluster for its function as a global transcription regulator in Escherichia coli when oxygen becomes scarce, To define the oxidation state and type of Fe-S cluster present in the active form of FNR, we have studied anaerobically purified FNR with Mossbauer spectroscopy, Our data showed that this form of FNR contained a [4Fe-4S](2+) cluster (delta = 0.45 mm/s; Delta E-Q = 1.22 mm/s) and that the [4Fe-4S](2+) cluster was rapidly destroyed on exposure of FNR to air. Under these conditions, the yellow-green active form of FNR turned deep red; analysis of sulfide indicated that 70% of the labile sulfide was still present, suggesting that the Fe-S cluster had been converted into a different form, Little [3Fe-4S] cluster was, however, detected by EPR. According to Mossbauer spectroscopy, the [4Fe-4S](2+) cluster was converted in about 60% yield to a [2Fe-2S](2+) cluster (delta = 0.28 mm/s; Delta E-Q = 0.58 mm/s) following 17 min of exposure to air, The [2Fe-2S](2+) cluster form of FNR was much more stable to oxygen, but was unable to sustain biological activity (e.g., DNA binding), However, DNA binding and the absorption spectrum characteristic of the [4Fe-4S](2+) cluster could be largely restored from the [2Fe-2S](2+) form when Cys, Fe, DTT, and the NifS protein were added. It has yet to be determined whether the form of FNR containing the [ZFe-2S](2+) cluster has any biological significance, e.g., as an in vivo intermediate that is more rapidly converted to the active form than the apoprotein.