Cytochrome C552 from Thermus thermophilus Engineered for Facile Substitution of Prosthetic Group
Cytochrome C552 from Thermus thermophilus Engineered for Facile Substitution of Prosthetic Group
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DOI：
10.1021/bi201048e
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发表时间：
2011-11-15
期刊：
影响因子：
2.9
通讯作者：
Watanabe, Yoshihito
中科院分区：
文献类型：
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作者：
Ibrahim, Sk Md;Nakajima, Hiroshi;Watanabe, Yoshihito
由于半胱氨酸残基和血红素之间的两个硫醚键，用非天然修复体基团简单地取代细胞色素C中的血红素C是很难实现的。菲等人的研究成果。证明了嗜热嗜热菌的细胞色素C-552在大肠杆菌的胞浆中大量生产，在血红素和Cys11之间具有可通过加热切割的共价键，并且与Cys14具有硫醚键[Fee，J.A.(2004)BioChemical 43,12162-12176]。在这一结果的提示下，我们准备了一个C14A突变体，预计突变体中的血红素物种只通过可热切割的键与多肽结合；因此，加热蛋白质后去除血红素是可行的。与预期相反，纯化后立即的C14A(纯化后的C14A)没有共价键。使用传统的酸丁酮方法提取亚铁血红素的尝试失败了，因为亚铁血红素和多肽之间形成了快速的连接。光谱分析表明，纯化后的C14A具有血红素b衍生物，其中两个外围乙烯基团中的一个已被含有活性羰基的基团取代。纯化后的C14A与[BH3CN](-)反应阻断了羰基上的键形成，从而定量地合成了不含血红素的apo-C14A。用铁和亚铁血红素b和锌原卟啉实现apo-C14A的重组。所有重组的C14A都有自发的共价键形成。我们认为，C14A是一个潜在的来源，以方便地生产人造细胞色素C，含有一个非天然的修复基。
The facile replacement of heme c in cytochromes c with non-natural prosthetic groups has been difficult to achieve due to two thioether linkages between cysteine residues and the heme. Fee et al. demonstrated that cytochrome C-552 from Thermus thermophilus, overproduced in the cytosol of E. coli, has a covalent linkage cleavable by heat between the heme and Cys11, as well as possessing the thioether linkage with Cys14 [Fee, J. A. (2004) Biochemistry 43, 12162-12176]. Prompted by this result, we prepared a C14A mutant, anticipating that the heme species in the mutant was bound to the polypeptide solely through the thermally cleavable linkage; therefore, the removal of the heme would be feasible after heating the protein. Contrary to this expectation, C14A immediately after purification (as-purified C14A) possessed no covalent linkage. An attempt to extract the heme using a conventional acid butanone method was unsuccessful due to rapid linkage formation between the heme and polypeptide. Spectroscopic analyses suggested that the as-purified C14A possessed a heme b derivative where one of two peripheral vinyl groups had been replaced with a group containing a reactive carbonyl. A reaction of the as-purified C14A with [BH3CN](-) blocked the linkage formation on the carbonyl group, allowing a quantitative yield of heme-free apo-C14A. Reconstitution of apo-C14A was achieved with ferric and ferrous heme b and zinc protoporphyrin. All reconstituted C14As showed spontaneous covalent linkage formation. We propose that C14A is a potential source for the facile production of an artificial cytochrome c, containing a non-natural prosthetic group.