Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, IN 47405, USA.
Mol Microbiol. 2012 Aug;85(4):734-46. doi: 10.1111/j.1365-2958.2012.08135.x. Epub 2012 Jul 16.
CrtJ from Rhodobacter capsulatus is a regulator of genes involved in the biosynthesis of haem, bacteriochlorophyll, carotenoids as well as structural proteins of the light harvesting-II complex. Fluorescence anisotropy-based DNA-binding analysis demonstrates that oxidized CrtJ exhibits ~20-fold increase in binding affinity over that of reduced CrtJ. Liquid chromatography electrospray tandem ionization mass spectrometric analysis using DAz-2, a sulfenic acid (-SOH)-specific probe, demonstrates that exposure of CrtJ to oxygen or to hydrogen peroxide leads to significant accumulation of a sulfenic acid derivative of Cys420 which is located in the helix-turn-helix (HTH) motif. In vivo labelling with 4-(3-azidopropyl)cyclohexane-1,3-dione (DAz-2) shows that Cys420 also forms a sulfenic acid modification in vivo when cells are exposed to oxygen. Moreover, a Cys420 to Ala mutation leads to a ~60-fold reduction of DNA binding activity while a Cys to Ser substitution at position 420 that mimics a cysteine sulfenic acid results in a ~4-fold increase in DNA binding activity. These results provide the first example where sulfenic acid oxidation of a cysteine in a HTH-motif leads to differential effects on gene expression.
来自荚膜红细菌的 CrtJ 是参与血红素、细菌叶绿素、类胡萝卜素以及光捕获 II 复合物结构蛋白生物合成的基因的调节剂。基于荧光各向异性的 DNA 结合分析表明，氧化的 CrtJ 对 DNA 的结合亲和力比还原的 CrtJ 高约 20 倍。使用 DAz-2（一种亚磺酸（-SOH）特异性探针）的液相色谱电喷雾串联电离质谱分析表明，CrtJ 暴露于氧气或过氧化氢会导致位于螺旋-转角-螺旋（HTH）基序中的 Cys420 的亚磺酸衍生物的大量积累。用 4-(3-叠氮丙基)环己烷-1,3-二酮 (DAz-2) 进行体内标记表明，当细胞暴露于氧气时，Cys420 也会在体内形成亚磺酸修饰。此外，Cys420 到 Ala 的突变导致 DNA 结合活性降低约 60 倍，而在位置 420 模拟半胱氨酸亚磺酸的 Cys 到 Ser 取代导致 DNA 结合活性增加约 4 倍。这些结果提供了第一个亚磺酸氧化 HTH 基序中的半胱氨酸导致基因表达产生差异影响的例子。
