Activation of respiratory Complex I from Escherichia coli studied by fluorescent probes

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• 作者：Nikolai Belevich^a ; Galina Belevich^a ; Zhiyong Chen^b ; ¹ ; Subhash C. Sinha^b ; ² ; Marina Verkhovskaya^a ; ^{Marina.Verkhovskaya@Helsinki.Fi}
• 关键词：Biological Sciences ; Biochemistry ; Biophysics
• 刊名：Heliyon
• 出版年：2017
• 出版时间：January 2017
• 年：2017
• 卷：3
• 期：1
• 全文大小：2673 K
• 卷排序：3

文摘

Respiratory Complex I from E. coli may exist in two interconverting forms: resting (R) and active (A). The R/A transition of purified, solubilized Complex I occurring upon turnover was studied employing two different fluorescent probes, Annine 6+, and NDB-acetogenin. NADH-induced fluorescent changes of both dyes bound to solubilized Complex I from E. coli were characterized as a function of the protein:dye ratio, temperature, ubiquinone redox state and the enzyme activity. Analysis of this data combined with time-resolved optical measurements of Complex I activity and spectral changes indicated two ubiquinone-binding sites; a possibility of reduction of the tightly-bound quinone in the resting state and reduction of the loosely-bound quinone in the active state is discussed. The results also indicate that upon the activation Complex I undergoes conformational changes which can be mapped to the junction of the hydrophilic and membrane domains in the region of the assumed acetogenin-binding site.