Site-Specific Chemical Labeling of Mitochondrial Respiratory Complex I through Ligand-Directed Tosylate Chemistry

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• 作者：Takahiro Masuya ; Masatoshi Murai ; Kentaro Ifuku ; Hironobu Morisaka ; Hideto Miyoshi
• 刊名：Biochemistry
• 出版年：2014
• 出版时间：April 15, 2014
• 年：2014
• 卷：53
• 期：14
• 页码：2307-2317
• 全文大小：495K
• 年卷期：v.53,no.14(April 15, 2014)
• ISSN：1520-4995

文摘

The site-specific chemical modification of NADH-quinone oxidoreductase (complex I) by various functional probes such as fluorophores and microbeads, without affecting the enzyme activity, may allow single-molecule analyses of putative dynamic conformational changes in the enzyme. In an attempt to address this challenge, we performed site-specific alkynylation of complex I in bovine heart submitochondrial particles by means of a ligand-directed tosylate (LDT) chemistry strategy with synthetic acetogenin ligand 1, which has an alkynylated tosylate in the tail moiety, as a high-affinity ligand against the enzyme. The terminal alkyne was chosen as the tag to be incorporated into the enzyme because this functional group can serve as a 鈥渇ooting鈥?for subsequent diverse chemical modifications via so-called click chemistry (i.e., azide鈥揳lkyne [3+2] cycloaddition in water). To identify the position alkynylated by ligand 1, fluorescent tetramethylrhodamine was covalently attached to the incorporated alkyne by click chemistry after the solubilization of complex I. Detailed proteomic analyses revealed that alkynylation occurred at Asp160 in the 49 kDa subunit, which may be located in the inner part of the putative quinone-binding cavity. The alkynylation was completely suppressed in the presence of an excess of other inhibitors such as bullatacin and quinazoline. While the reaction yield of the alkynylation step via LDT chemistry was estimated to be 50%, the alkynylation unfortunately resulted in the almost complete inhibition of enzyme activity. Nevertheless, the results of this study demonstrate that complex I can be site-specifically alkynylated through LDT chemistry, providing a clue about the diverse chemical modifications of the enzyme in combination with click chemistry.