Centre for Molecular and Structural Biochemistry, School of Chemistry, University of East Anglia, Norwich NR4 7TJ, UK.
Biochemistry. 2009 Dec 29;48(51):12252-64. doi: 10.1021/bi901498v.
WhiD, a member of the WhiB-like (Wbl) family of iron-sulfur proteins found exclusively within the actinomycetes, is required for the late stages of sporulation in Streptomyces coelicolor. Like all other Wbl proteins, WhiD has not so far been purified in a soluble form that contains a significant amount of cluster, and characterization has relied on cluster-reconstituted protein. Thus, a major goal in Wbl research is to obtain and characterize native protein containing iron-sulfur clusters. Here we report the analysis of S. coelicolor WhiD purified anaerobically from Escherichia coli as a soluble protein containing a single 4Fe-4S cluster ligated by four cysteines. Upon exposure to oxygen, spectral features associated with the [4Fe-4S] cluster were lost in a slow reaction that unusually yielded apo-WhiD directly without significant concentrations of cluster intermediates. This process was found to be highly pH dependent with an optimal stability observed between pH 7.0 and pH 8.0. Low molecular weight thiols, including a mycothiol analogue and thioredoxin, exerted a small but significant protective effect against WhiD cluster loss, an activity that could be of physiological importance. 4Fe-4S WhiD was found to react much more rapidly with superoxide than with either oxygen or hydrogen peroxide, which may also be of physiological significance. Loss of the [4Fe-4S] cluster to form apoprotein destabilized the protein fold significantly but did not lead to complete unfolding. Finally, apo-WhiD exhibited negligible activity in an insulin-based disulfide reductase assay, demonstrating that it does not function as a general protein disulfide reductase.
WhiD 是放线菌中特有的 WhiB 样（Wbl）家族的铁硫蛋白成员，它是链霉菌属（Streptomyces coelicolor）孢子形成后期所必需的。与所有其他 Wbl 蛋白一样，WhiD 迄今尚未以含有大量簇的可溶形式被纯化，并且其特性依赖于重新构成簇的蛋白质。因此，Wbl 研究的主要目标是获得和表征含有铁硫簇的天然蛋白。在这里，我们报道了从大肠杆菌中厌氧纯化的链霉菌属 WhiD 的分析，该蛋白作为一种可溶性蛋白，含有一个由四个半胱氨酸连接的单一4Fe-4S簇。暴露于氧气后，与[4Fe-4S]簇相关的光谱特征在缓慢反应中丢失，该反应异常直接产生无配体的 WhiD，而没有明显的簇中间体浓度。该过程高度依赖于 pH 值，在 pH 值 7.0 到 pH 值 8.0 之间观察到最佳稳定性。低分子量硫醇，包括一种类麦角硫因和硫氧还蛋白，对 WhiD 簇的丢失具有微小但显著的保护作用，这种活性可能具有生理重要性。发现4Fe-4S WhiD 与超氧化物的反应速度比与氧气或过氧化氢的反应速度快得多，这也可能具有生理意义。[4Fe-4S]簇的丢失导致脱辅基蛋白的蛋白质折叠显著失稳，但不会导致完全展开。最后，apo-WhiD 在基于胰岛素的二硫键还原酶测定中表现出可忽略不计的活性，表明它不能作为一般的蛋白质二硫键还原酶发挥作用。
