[2Fe-2S]-ferredoxin binds directly to cysteine desulfurase and supplies an electron for iron-sulfur cluster assembly but is displaced by the scaffold protein or bacterial frataxin.
[2Fe-2S]-ferredoxin binds directly to cysteine desulfurase and supplies an electron for iron-sulfur cluster assembly but is displaced by the scaffold protein or bacterial frataxin.
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DOI：
10.1021/ja401950a
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发表时间：
2013-06-05
影响因子：
15
通讯作者：
Markley, John L.
中科院分区：
文献类型：
--
作者：
Kim, Jin Hae;Frederick, Ronnie O.;Reinen, Nichole M.;Troupis, Andrew T.;Markley, John L.
大肠杆菌[2Fe-2S]-铁氧还蛋白(FDX)是由ISC操纵子和其他参与铁-硫团簇生物发生管家机制的蛋白质共同编码的。尽管有人提出FDx提供电子将半胱氨酸脱硫酶(Iscs)产生的硫烷硫(S0)还原为硫化物(S2-)，这是Fe-S簇在支架蛋白(IscU)上组装所必需的，但还缺乏直接的实验证据来证明FDx的作用。在这里，我们证明了FDX(在任一氧化状态下)与ISCS直接相互作用。发现FDX上的相互作用面包括接近其Fe-S簇的残基。此外，ISCS的C328残基在氧化剂存在下与FDX形成二硫键。C328是已知的从ISCS活性中心吸收硫并将其输送到ISCU的半胱氨酸残基的残基。只有在L-半胱氨酸存在的情况下，还原FDX的电子才转移到ISCS，而不是C328S突变体。我们发现FDX、ISCU和CyaY(细菌的Frataxin)竞争ISCS上重叠的结合位点。这种互斥解释了CyaY抑制Fe-S簇生物发生的机制。这些结果(1)表明还原的FDX为ISCS复合体提供一个电子，因为S0是由Cys酶促转化为Ala而产生的；(2)解释了FDX作为ISC操纵子成员的作用。
Escherichia coli [2Fe-2S]-ferredoxin (Fdx) is encoded by the isc operon along with other proteins involved in the ‘house-keeping’ mechanism of iron–sulfur cluster biogenesis. Although it has been proposed that Fdx supplies electrons to reduce sulfane sulfur (S0) produced by the cysteine desulfurase (IscS) to sulfide (S2–) as required for the assembly of Fe–S clusters on the scaffold protein (IscU), direct experimental evidence for the role of Fdx has been lacking. Here, we show that Fdx (in either oxidation state) interacts directly with IscS. The interaction face on Fdx was found to include residues close to its Fe–S cluster. In addition, C328 of IscS, the residue known to pick up sulfur from the active site of IscS and deliver it to the Cys residues of IscU, formed a disulfide bridge with Fdx in the presence of an oxidizing agent. Electrons from reduced Fdx were transferred to IscS only in the presence of l-cysteine, but not to the C328S variant. We found that Fdx, IscU, and CyaY (the bacterial frataxin) compete for overlapping binding sites on IscS. This mutual exclusion explains the mechanism by which CyaY inhibits Fe–S cluster biogenesis. These results (1) show that reduced Fdx supplies one electron to the IscS complex as S0 is produced by the enzymatic conversion of Cys to Ala and (2) explain the role of Fdx as a member of the isc operon.
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