除了 [Fe2S2]2+ 簇之外，单硫醇谷氧还蛋白可以结合线性 [Fe3S4]+ 和 [Fe4S4]2+ 簇：光谱表征和功能影响,Journal of the American Chemical Society

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除了 [Fe2S2]2+ 簇之外，单硫醇谷氧还蛋白可以结合线性 [Fe3S4]+ 和 [Fe4S4]2+ 簇：光谱表征和功能影响
Journal of the American Chemical Society ( IF 14.4 ) Pub Date : 2013-09-26 , DOI: 10.1021/ja407059n
Bo Zhang ₁ , Sibali Bandyopadhyay , Priyanka Shakamuri , Sunil G Naik , Boi Hanh Huynh , Jérémy Couturier , Nicolas Rouhier , Michael K Johnson

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酿酒酵母线粒体谷氧还蛋白 5 (Grx5) 是无处不在的单硫醇谷氧还蛋白类的原型成员，具有严格保守的 CGFS 活性位点序列，已被证明在生物 [Fe2S2](2+) 集群贩运中发挥作用。在这项工作中，我们展示了重组酿酒酵母 Grx5 在无细胞提取物长时间暴露于空气中或在谷胱甘肽存在下进行厌氧重建后有氧纯化，主要包含线性 [Fe3S4](+) 簇。线性 [Fe3S4](+) 簇的激发态电子特性和基态电子和振动特性已使用 UV-vis 吸收/CD/MCD、EPR、穆斯堡尔和共振拉曼光谱进行表征。结果揭示了菱形 S = 5/2 线性 [Fe3S4](+) 簇，其性质类似于合成线性 [Fe3S4](+) 簇和紫色乌头酸酶中的线性 [Fe3S4](+) 簇。此外，结果表明，先前报道的许多单硫醇 Grx 的 Fe-S 簇含量被完全误解为 [Fe2S2](2+) 簇，而不是线性 [Fe3S4](+) 簇或线性 [Fe3S4 ](+) 和 [Fe2S2](2+) 簇。在没有 GSH 的情况下，Grx5 的厌氧重组产生一种二聚体形式，其中包含一个 [Fe4S4](2+) 簇，通过完整的簇转移能够在体外激活脱辅基乌头酸酶。Grx5 中线性 [Fe3S4](+) 和 [Fe4S4](2+) 簇的连接已通过光谱、突变和分析研究进行评估。

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Monothiol Glutaredoxins Can Bind Linear [Fe3S4]+and [Fe4S4]2+Clusters in Addition to [Fe2S2]2+Clusters: Spectroscopic Characterization and Functional Implications

Saccharomyces cerevisiae mitochondrial glutaredoxin 5 (Grx5) is the archetypical member of a ubiquitous class of monothiol glutaredoxins with a strictly conserved CGFS active-site sequence that has been shown to function in biological [Fe2S2](2+) cluster trafficking. In this work, we show that recombinant S. cerevisiae Grx5 purified aerobically, after prolonged exposure of the cell-free extract to air or after anaerobic reconstitution in the presence of glutathione, predominantly contains a linear [Fe3S4](+) cluster. The excited-state electronic properties and ground-state electronic and vibrational properties of the linear [Fe3S4](+) cluster have been characterized using UV-vis absorption/CD/MCD, EPR, Mössbauer, and resonance Raman spectroscopies. The results reveal a rhombic S = 5/2 linear [Fe3S4](+) cluster with properties similar to those reported for synthetic linear [Fe3S4](+) clusters and the linear [Fe3S4](+) clusters in purple aconitase. Moreover, the results indicate that the Fe-S cluster content previously reported for many monothiol Grxs has been misinterpreted exclusively in terms of [Fe2S2](2+) clusters, rather than linear [Fe3S4](+) clusters or mixtures of linear [Fe3S4](+) and [Fe2S2](2+) clusters. In the absence of GSH, anaerobic reconstitution of Grx5 yields a dimeric form containing one [Fe4S4](2+) cluster that is competent for in vitro activation of apo-aconitase, via intact cluster transfer. The ligation of the linear [Fe3S4](+) and [Fe4S4](2+) clusters in Grx5 has been assessed by spectroscopic, mutational, and analytical studies. Potential roles for monothiol Grx5 in scavenging and recycling linear [Fe3S4](+) clusters released during protein unfolding under oxidative stress conditions and in maturation of [Fe4S4](2+) cluster-containing proteins are discussed in light of these results.

更新日期：2013-09-26

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