Frataxin Directly Stimulates Mitochondrial Cysteine Desulfurase by Exposing Substrate-binding Sites, and a Mutant Fe-S Cluster Scaffold Protein with Frataxin-bypassing Ability Acts Similarly
Frataxin Directly Stimulates Mitochondrial Cysteine Desulfurase by Exposing Substrate-binding Sites, and a Mutant Fe-S Cluster Scaffold Protein with Frataxin-bypassing Ability Acts Similarly
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DOI：
10.1074/jbc.m113.525857
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发表时间：
2013-12-27
影响因子：
4.8
通讯作者：
Pain, Debkumar
中科院分区：
文献类型：
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作者：
Pandey, Alok;Gordon, Donna M.;Pain, Debkumar
对于线粒体中的铁硫 (Fe-S) 簇合成，硫是通过 Nfs1 的半胱氨酸脱硫酶活性从氨基酸半胱氨酸衍生而来。该酶与含磷酸吡哆醛位点的底物半胱氨酸结合，并以取决于辅助蛋白 Isd11 的方式在活性位点半胱氨酸上形成过硫化物。然后过硫化物被转移到支架 Isu，在那里与铁结合形成 Fe-S 簇中间体。 Frataxin 参与了这一过程，但尚不清楚在何处以及如何参与，而且缺乏会导致弗里德赖希共济失调。使用纯化的蛋白质和分离的线粒体，我们在此表明酵母 frataxin 同源物 (Yfh1) 通过暴露底物结合位点直接、特异性地刺激半胱氨酸与 Nfs1 的结合。 frataxin 的这一新功能不需要铁、Isu1 或 Isd11。一旦与 Nfs1 结合，底物半胱氨酸就是 Nfs1 过硫化物的来源，但此步骤独立于 frataxin，并严格依赖于 Isd11。最近，发现 Isu1 的点突变可以绕过许多 frataxin 功能。这里提供的数据表明 Isu1 抑制器模仿 frataxin 对 Nfs1 的影响，解释了绕过活动。我们提出了 Nfs1 过硫化物形成活性的调节机制。具体来说，为了获得最佳活性，酶中必须发生至少两种单独的构象变化，如下所示：一种是由 frataxin 相互作用介导的，暴露埋藏的底物结合位点，另一种是由 Isd11 相互作用介导的，该相互作用使结合的底物半胱氨酸和活性位点半胱氨酸接近以形成过硫化物。
For iron-sulfur (Fe-S) cluster synthesis in mitochondria, the sulfur is derived from the amino acid cysteine by the cysteine desulfurase activity of Nfs1. The enzyme binds the substrate cysteine in the pyridoxal phosphate-containing site, and a persulfide is formed on the active site cysteine in a manner depending on the accessory protein Isd11. The persulfide is then transferred to the scaffold Isu, where it combines with iron to form the Fe-S cluster intermediate. Frataxin is implicated in the process, although it is unclear where and how, and deficiency causes Friedreich ataxia. Using purified proteins and isolated mitochondria, we show here that the yeast frataxin homolog (Yfh1) directly and specifically stimulates cysteine binding to Nfs1 by exposing substrate-binding sites. This novel function of frataxin does not require iron, Isu1, or Isd11. Once bound to Nfs1, the substrate cysteine is the source of the Nfs1 persulfide, but this step is independent of frataxin and strictly dependent on Isd11. Recently, a point mutation in Isu1 was found to bypass many frataxin functions. The data presented here show that the Isu1 suppressor mimics the frataxin effects on Nfs1, explaining the bypassing activity. We propose a regulatory mechanism for the Nfs1 persulfide-forming activity. Specifically, at least two separate conformational changes must occur in the enzyme for optimum activity as follows: one is mediated by frataxin interaction that exposes the buried substrate-binding sites, and the other is mediated by Isd11 interaction that brings the bound substrate cysteine and the active site cysteine in proximity for persulfide formation.