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

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Abstract
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.
Funding Information
  • Leibniz Association
  • German Academic Exchange Service
  • Deutsche Forschungsgemeinschaft
  • Alexander von Humboldt-Stiftung
  • Fundação para a Ciência e a Tecnologia (PD/BD/113985/2015, PTDC/BBB-BQB/2294/2012, UID/CBQ/04612/2013)
  • Nederlandse Organisatie voor Wetenschappelijk Onderzoek (722.011.003)

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