Characterization of the Bradyrhizobium japonicum CycY protein, a membrane-anchored periplasmic thioredoxin that may play a role as a reductant in the biogenesis of c-type cytochromes
Characterization of the Bradyrhizobium japonicum CycY protein, a membrane-anchored periplasmic thioredoxin that may play a role as a reductant in the biogenesis of c-type cytochromes
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DOI：
10.1074/jbc.272.7.4467
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发表时间：
1997-02-14
影响因子：
4.8
通讯作者：
ThonyMeyer, L
中科院分区：
文献类型：
--
作者：
Fabianek, RA;HuberWunderlich, M;ThonyMeyer, L
在慢生根瘤菌中发现了一个新的膜锚定周质硫氧还蛋白样蛋白。它是cycY的产物，cycY是细胞色素c生物合成基因簇中的最后一个基因。突变分析表明，cycY是必不可少的生物合成的所有C型细胞色素在这种细菌。CycY蛋白被证明是出口到周质的N-末端信号序列样结构域。来自膜和可溶性级分的Western印迹分析的结果表明，CycY蛋白保持与膜结合。在大肠杆菌中表达缺乏其N-末端膜锚（CycY*）的蛋白质的可溶性版本，并从周质部分纯化至均一。该蛋白表现出氧化还原反应性和性质类似于其他硫氧还蛋白，如荧光淬灭的氧化形式。其与谷胱甘肽的平衡常数确定为168 mM，由此计算出标准氧化还原电位为-0.217 V，这表明CycY可能在周质的氧化环境中充当还原剂。这与我们的假设，即CycY是必需的，直接或间接地，减少血红素结合位点半胱氨酸在C型脱辅基色素的CXXCH基序前血红素附件发生。
A new member of membrane-anchored periplasmic thioredoxin-like proteins was identified in Bradyrhizobium japonicum. It is the product of cycY, the last gene in a cluster of cytochrome c biogenesis genes. Mutational analysis revealed that cycY is essential for the biosynthesis of all c-type cytochromes in this bacterium. The CycY protein was shown to be exported to the periplasm by its N-terminal signal sequence-like domain. Results from Western blot analyses of membrane and soluble fractions indicated that the CycY protein remains bound to the membrane. A soluble version of the protein devoid of its N-terminal membrane anchor (CycY*) was expressed in Escherichia coli and purified to homogeneity from the periplasmic fraction. The protein showed redox reactivity and properties similar to other thioredoxins such as fluorescence quenching in the oxidized form. Its equilibrium constant with glutathione was determined to be 168 mM, from which a standard redox potential of -0.217 V was calculated, suggesting that CycY might act as a reductant in the otherwise oxidative environment of the periplasm. This is in agreement with our hypothesis that CycY is required, directly or indirectly, for the reduction of the heme-binding site cysteines in the CXXCH motif of c-type apocytochromes before heme attachment occurs.