Substrate–Protein Interactions of Type II NADH:Quinone Oxidoreductase from <i>Escherichia coli</i>

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 <i>Escherichia coli</i> (<i>Ec</i>NDH-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 <i>Ec</i>NDH-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

Reconstitution of Respiratory Complex I on a Biomimetic Membrane Supported on Gold Electrodes

For the first time, respiratory complex I has been reconstituted on an electrode preserving its structure and activity. Respiratory complex I is a membrane-bound enzyme that has an essential function in cellular energy production. It couples NADH:quinone oxidoreduction to translocation of ions across the cellular (in prokaryotes) or mitochondrial membranes. Therefore, complex I contributes to the establishment and maintenance of the transmembrane difference of electrochemical potential required for adenosine triphosphate synthesis, transport, and motility. Our new strategy has been applied for reconstituting the bacterial complex I from Rhodothermus marinus onto a biomimetic membrane supported on gold electrodes modified with a thiol self-assembled monolayer (SAM). Atomic force microscopy and faradaic impedance measurements give evidence of the biomimetic construction, whereas electrochemical measurements show its functionality. Both electron transfer and proton translocation by respiratory complex I were monitored, simulating in vivo conditions

Catalytic Activity and Proton Translocation of Reconstituted Respiratory Complex I Monitored by Surface-Enhanced Infrared Absorption Spectroscopy

Respiratory complex I (CpI) is a key player in the way organisms obtain energy, being an energy transducer, which couples nicotinamide adenine dinucleotide (NADH)/quinone oxidoreduction with proton translocation by a mechanism that remains elusive so far. In this work, we monitored the function of CpI in a biomimetic, supported lipid membrane system assembled on a 4-aminothiophenol (4-ATP) self-assembled monolayer by surface-enhanced infrared absorption spectroscopy. 4-ATP serves not only as a linker molecule to a nanostructured gold surface but also as pH sensor, as indicated by concomitant density functional theory calculations. In this way, we were able to monitor NADH/quinone oxidoreduction-induced transmembrane proton translocation via the protonation state of 4-ATP, depending on the net orientation of CpI molecules induced by two complementary approaches. An associated change of the amide I/amide II band intensity ratio indicates conformational modifications upon catalysis which may involve movements of transmembrane helices or other secondary structural elements, as suggested in the literature [Di Luca , Proc. Natl. Acad. Sci. U.S.A., 2017, 114, E6314−E6321]