Substrate–Protein Interactions of Type II NADH:Quinone Oxidoreductase from Escherichia coli

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• 作者：Johannes Salewski ; Ana P. Batista ; Filipa V. Sena ; Diego Millo ; Ingo Zebger ; Manuela M. Pereira ; Peter Hildebrandt
• 刊名：Biochemistry
• 出版年：2016
• 出版时间：May 17, 2016
• 年：2016
• 卷：55
• 期：19
• 页码：2722-2734
• 全文大小：637K
• 年卷期：0
• ISSN：1520-4995

文摘

Type II NADH:quinone oxidoreductases (NDH-2s) are membrane proteins involved in respiratory chains and responsible for the maintenance of NADH/NAD⁺ balance in cells. NDH-2s are the only enzymes with NADH dehydrogenase activity present in the respiratory chain of many pathogens, and thus, they were proposed as suitable targets for antimicrobial therapies. In addition, NDH-2s were also considered key players for the treatment of complex I-related neurodegenerative disorders. In this work, we explored substrate–protein interaction in NDH-2 from Escherichia coli (EcNDH-2) combining surface-enhanced infrared absorption spectroscopic studies with electrochemical experiments, fluorescence spectroscopy assays, and quantum chemical calculations. Because of the specific stabilization of substrate complexes of EcNDH-2 immobilized on electrodes, it was possible to demonstrate the presence of two distinct substrate binding sites for NADH and the quinone and to identify a bound semiprotonated quinol as a catalytic intermediate.