The Escherichia coli cytochrome c maturation (Ccm) system does not detectably attach heme to single cysteine variants of an apocytochrom c
The Escherichia coli cytochrome c maturation (Ccm) system does not detectably attach heme to single cysteine variants of an apocytochrom c
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DOI：
10.1074/jbc.m204963200
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发表时间：
2002-09-13
影响因子：
4.8
通讯作者：
Ferguson, SJ
中科院分区：
文献类型：
--
作者：
Allen, JWA;Tomlinson, EJ;Ferguson, SJ
细胞色素c的典型特征在于血红素通过两个硫醚键与Cys-Xaa-Xaa-Cys-His肽基序的半胱氨酸残基共价连接至多肽。在许多革兰氏阴性细菌中，血红素通过周质功能的细胞色素c成熟（Ccm）蛋白附着于多肽。例外的是，嗜热氢杆菌细胞色素c（552），它有一个正常的CXXCH血红素结合基序，和AXXCH，CXXAH和AXXAH基序的变体，可以在大肠杆菌的细胞质中表达为稳定的全细胞色素。通过使用信号肽将这些蛋白质靶向到周质，有或没有Cent蛋白质的共表达，我们评估了Ccm系统将血红素连接到血红素结合基序中没有、一个或两个半胱氨酸残基的蛋白质的能力。只有野生型蛋白质，与两个半胱氨酸，有效地处理，从而积累在周质中作为一个holocytochrome。这是涉及脱辅基细胞色素c的两个半胱氨酸残基作为Ccm型革兰氏阴性细菌细胞色素c生物合成中的中间体的二硫键形成和/或仅一对半胱氨酸可以被血红素附着装置识别的有力证据。
Cytochromes c are typically characterized by the covalent attachment of heme to polypeptide through two thioether bonds with the cysteine residues of a Cys-Xaa-Xaa-Cys-His peptide motif. In many Gram-negative bacteria, the heme is attached to the polypeptide by the periplasmically functioning cytochrome c maturation (Ccm) proteins. Exceptionally, Hydrogenobacter thermophilus cytochrome c(552), which has a normal CXXCH heme-binding motif, and variants with AXXCH, CXXAH, and AXXAH motifs, can be expressed as stable holocytochromes in the cytoplasm of Escherichia coli. By targeting these proteins to the periplasm using a signal peptide, with or without co-expression of the Cent proteins, we have assessed the ability of the Ccm system to attach heme to proteins with no, one, or two cysteine residues in the heme-binding motif. Only the wild-type protein, with two cysteines, was effectively processed and thus accumulated in the periplasm as a holocytochrome. This is strong evidence for disulfide bond formation involving the two cysteine residues of apocytochrome c as an intermediate in Ccm-type Gram-negative bacterial cytochrome c biogenesis and/or that only a pair of cysteines can be recognized by the heme attachment apparatus.