A thioreduction pathway tethered to the membrane for periplasmic cytochromes c biogenesis; in vitro and in vivo studies
A thioreduction pathway tethered to the membrane for periplasmic cytochromes c biogenesis; in vitro and in vivo studies
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DOI：
10.1006/jmbi.1997.1227
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发表时间：
1997-09-05
影响因子：
5.6
通讯作者：
Kranz, RG
中科院分区：
文献类型：
--
作者：
Monika, EM;Goldman, BS;Kranz, RG
C型细胞色素与其他血红素蛋白的区别在于两个血红素乙烯基与脱辅基蛋白上的两个半胱氨酸残基（在CXXCH结构域）的共价连接。本研究旨在阐明革兰氏阴性菌Rhodobacter capsulatus中细胞色素c生物合成所需的两种蛋白质HelX和Ccl 2的作用和拓扑位置。从它们的一级序列来看，这些蛋白质中的每一个都具有CXXC基序，该基序可能参与脱辅基细胞色素c的半胱氨酸残基的还原，这是共价连接到血红素的先决条件。HelX和Ccl 2的定点诱变的结果表明，每个半胱氨酸残基是所需的蛋白质的体内功能。我们证明了R.荚膜通过其未切割的信号序列与细胞质膜相连。Ccl 2通过存在于C末端的单个跨膜结构域与周质中蛋白质的N末端三分之二连接。因此，两个CXXC基序都暴露于周质。过量产生完整的HelX蛋白和Ccl 2的可溶性N-末端部分（称为Ccl 2 *）并从周质级分纯化。有效地处理Ccl 2 * 信号序列。用这些纯化的蛋白质进行的体外研究表明，虽然两者都不能还原胰岛素，但HelX可以还原Ccl 2半胱氨酸残基，并且Ccl 2半胱氨酸残基被含有CXXCH结构域的脱辅基细胞色素c肽氧化。一个helX缺失突变体的回复突变体被分离，重新获得的能力，使C型细胞色素（从而光合生长），这些抑制菌株中的一些增强光合生长的添加硫还原剂。相反，ccl 2缺失菌株的回复突变体在任何条件下都不能分离。这些结果表明，HelX和Ccl 2蛋白形成硫代还原途径（HelX -> Ccl 2->脱辅基细胞色素c），由此Ccl 2功能可能对脱辅基细胞色素c高度特异，而HelX可能充当具有邻位半胱氨酸的蛋白质的更一般的还原剂。(C)出版社：Academic Press Limited。
The c-type cytochromes are distinguished from other heme proteins by the covalent Ligation of two heme vinyl groups to two cysteine residues on the apoprotein (at a CXXCH domain). The present study was undertaken to elucidate the roles and topological locations of two of the proteins necessary for cytochrome c biogenesis, the HelX and Ccl2 proteins in the Gram-negative bacteria Rhodobacter capsulatus. From their primary sequence, each of these proteins has a CXXC motif that could be involved in the reduction of the cysteine residues of the apocytochromes c, a prerequisite for covalent ligation to the heme. Results of site-directed mutagenesis of HelX and Ccl2 demonstrate that each cysteine residue is required for the in vivo function of the protein. We demonstrate that the native HelX in R. capsulatus is tethered to the cytoplasmic membrane via its uncleaved signal sequence. Ccl2 is tethered by a single transmembrane domain present in the C terminus with the N-terminal two-thirds of the protein in the periplasm. Thus, both CXXC motifs are exposed to the periplasm. The complete HelX protein and the soluble N-terminal portion of Ccl2 (called Ccl2*) were overproduced and purified from periplasmic fractions. The Ccl2* signal sequence is efficiently processed. In vitro studies with these purified proteins indicate that although neither can reduce insulin, HelX can reduce the Ccl2 cysteine residues and the Ccl2 cysteine residues are oxidized by an apocytochrome c peptide containing the CXXCH domain. Revertants of an helX deletion mutant were isolated that regain the ability to make c-type cytochromes (and thus grow photosynthetically); some of these suppressor strains are enhanced for photosynthetic growth by the addition of thio-reducing agents. In contrast, revertants of a ccl2 deletion strain could not be isolated under any condition. These results suggest that the HelX and Ccl2 proteins form a thioreduction pathway (HelX --> Ccl2 --> apocytochrome c) whereby Ccl2 function may be highly specific for apocytochromes c while HelX may act as a more general reductant of proteins with vicinal cysteines. (C) 1997 Academic Press Limited.