Photolabile ubiquinone analogues for identification and characterization of quinone binding sites in proteins
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- 作者：Zhichao Pei ; Tobias Gustavsson ; Robert Roth ; Torbjö ; rn Frejd ; Cecilia Hä ; gerhä ; ll
- 关键词：Azidoquinone synthesis ; NADH ; quinone oxidoreductase ; Succinate ; quinone oxidoreductase ; Flavodoxin ; DT-diaphorase ; NQO1 ; Tryptophane repressor binding protein WrbA ; Quinone binding sites ; Photolabeling ; MALDI-TOF
- 刊名：Bioorganic and Medicinal Chemistry
- 出版年：2010
- 出版时间：15 May 2010
- 年：2010
- 卷：18
- 期：10
- 页码：3457-3466
- 全文大小：507 K
文摘
Quinones are essential components in most cell and organelle bioenergetic processes both for direct electron and/or proton transfer reactions but also as means to regulate various bioenergetic processes by sensing cell redox states. To understand how quinones interact with proteins, it is important to have tools for identifying and characterizing quinone binding sites. In this work three different photo-reactive azidoquinones were synthesized, two of which are novel compounds, and the methods of synthesis was improved. The reactivity of the azidoquinones was first tested with model peptides, and the adducts formed were analyzed by mass spectrometry. The added mass detected was that of the respective azidoquinone minus N2. Subsequently, the biological activity of the three azidoquinones was assessed, using three enzyme systems of different complexity, and the ability of the compounds to inactivate the enzymes upon illumination with long wavelength UV light was investigated. The soluble flavodoxin-like protein WrbA could only use two of the azidoquinones as substrates, whereas respiratory chain Complexes I and II could utilize all three compounds as electron acceptors. Complex II, purified in detergent, was very sensitive to illumination also in the absence of azidoquinones, making the ‘therapeutic window’ in that enzyme rather narrow. In membrane bound Complex I, only two of the compounds inactivated the enzyme, whereas illumination in the presence of the third compound left enzyme activity essentially unchanged. Since unspecific labeling should be equally effective for all the compounds, this demonstrates that the observed inactivation is indeed caused by specific labeling.