Variant c-type cytochromes as probes of the substrate specificity of the E. coli cytochrome c maturation (Ccm) apparatus
Variant c-type cytochromes as probes of the substrate specificity of the E. coli cytochrome c maturation (Ccm) apparatus
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DOI：
10.1042/bj20081999
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发表时间：
2009-04-01
影响因子：
4.1
通讯作者：
Ferguson, Stuart J.
中科院分区：
文献类型：
--
作者：
Allen, James W. A.;Sawyer, Elizabeth B.;Ferguson, Stuart J.
C型细胞色素的特征通常在于铁辅因子血红素通过血红素的乙烯基和CXXCH（Cys-Xaa-Xaa-Cys-His）基序的巯基之间的两个硫醚键共价连接到蛋白质上。在细胞中，血红素附着是酶催化的翻译后修饰。我们先前已经证明，含有CXXCH血红素结合基序的大肠杆菌细胞色素B（562）变体与E. coli Ccm（细胞色素c成熟）蛋白导致正确形成的c型细胞色素的均匀成熟。相反，在没有Ccm装置的情况下，产品holocytochrome是异质的，主要物种具有血红素倒置反连接通过只有一个硫醚键。在本研究中，我们使用细胞色素的进一步变体来研究E。coli Ccm装置。该系统将成熟的C型细胞色素命名为CCXXCH、CCXCH、CXCCH和CXXCHC基序，尽管这些基序在自然界中并不存在，而且额外的半胱氨酸残基原则上可能会破坏必须与大肠杆菌中的二硫键氧化和还原蛋白复杂相互作用的生物合成蛋白。大肠杆菌周质。Ccm蛋白还可以将血红素连接到CXnCH类型的基序上，其中n的范围为2至6。对于n = 3和4，血红素连接是正确和均匀的，但对于较高的n值的holocytochromes显示与共价血红素连接过程相关的硫和/或氧原子的氧化加成。我们的意见的血红素附着反应，基因组分析和Ccm系统的底物特异性的影响，进行了讨论。
c-type cytochromes are normally characterized by covalent attachment of the iron cofactor haem to protein through two thioether bonds between the vinyl groups of the haem and the thiol groups of a CXXCH (Cys--Xaa-Xaa-Cys-His) motif. In cells, the haem attachment is an enzyme-catalysed post-translational modification. We have previously shown that co-expression of a variant of Escherichia coli cytochrome b(562) containing a CXXCH haem-binding motif with the E. coli Ccm (cytochrome c maturation) proteins resulted in homogeneous maturation of a correctly formed c-type cytochrome. In contrast, in the absence of the Ccm apparatus, the product holocytochrome was heterogeneous, the main species having haem inverted anti attached through only one thioether bond. In the present study We use further variants of cytochrome to investigate the substrate specificity of the E. coli Ccm apparatus. The system call mature c-type cytochromes with CCXXCH, CCXCH, CXCCH and CXXCHC motifs, even though these are not found naturally and the extra cysteine residue might, in principle, disrupt the biogenesis proteins which must interact intricately with disulfide-bond oxidizing and reducing proteins in the E. coli periplasm. The Ccm proteins can also attach haem to motifs of the type CXnCH where n ranges from 2 to 6. For n = 3 and 4, the haem attachment was correct and homogeneous, but for higher values of n the holocytochromes displayed oxidative addition of sulfur and/or oxygen atoms associated with the covalent haem-attachment process. The implications of our observations for the haem-attachment reaction, for genome analyses and for the substrate specificity of the Ccm system, are discussed.