Cytochrome c552 from Thermus thermophilus Engineered for Facile Substitution of Prosthetic Group

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• 作者：Sk. Md. Ibrahim ; Hiroshi Nakajima ; Takehiro Ohta ; Kalaivani Ramanathan ; Nobuyuki Takatani ; Yoshinori Naruta ; Yoshihito Watanabe
• 刊名：Biochemistry
• 出版年：2011
• 出版时间：November 15, 2011
• 年：2011
• 卷：50
• 期：45
• 页码：9826-9835
• 全文大小：404K
• 年卷期：v.50,no.45(November 15, 2011)
• ISSN：1520-4995

文摘

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 c₅₅₂ 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鈥?2176]. 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鈥揵utanone 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]^{鈭?/sup> 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.}