Substrate-Protein Interactions of Type II NADH:Quinone Oxidoreductase from Escherichia coli.
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.
Technische Universität Berlin , Institut für Chemie, Sekr. PC14, Straße des 17. Juni 135, D-10623 Berlin, Germany.
Johannes
J
Salewski
Instituto de Tecnologia Química e Biológica-António Xavier, Universidade Nova de Lisboa , Av. da República EAN, P-2780-157 Oeiras, Portugal.
Ana P
AP
Batista
Instituto de Tecnologia Química e Biológica-António Xavier, Universidade Nova de Lisboa , Av. da República EAN, P-2780-157 Oeiras, Portugal.
Filipa V
FV
Sena
Biomolecular Spectroscopy/LaserLaB Amsterdam, Vrije Universiteit Amsterdam , De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
Diego
D
Millo
Technische Universität Berlin , Institut für Chemie, Sekr. PC14, Straße des 17. Juni 135, D-10623 Berlin, Germany.
Ingo
I
Zebger
Instituto de Tecnologia Química e Biológica-António Xavier, Universidade Nova de Lisboa , Av. da República EAN, P-2780-157 Oeiras, Portugal.
Manuela M
MM
Pereira
Technische Universität Berlin , Institut für Chemie, Sekr. PC14, Straße des 17. Juni 135, D-10623 Berlin, Germany.
Peter
P
Hildebrandt
D016227:Benzoquinones / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009243:NAD / Q000737:chemistry*; D009245:NADH Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D016227:Benzoquinones; D029968:Escherichia coli Proteins; D009243:NAD; C004532:quinone; C088908:NADH dehydrogenase II; D009245:NADH Dehydrogenase