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

Abstract

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 ].

Surface-Enhanced Infrared Absorption Spectroscopy of Bacterial Nitric Oxide Reductase under Electrochemical Control Using a Vibrational Probe of Carbon Monoxide

“…To understand the reaction mechanism, electrochemical properties of the four iron cofactors of NORs have been studied using spectroelectrochemical titration − and protein film electrochemistry (PFE), , which provides current–potential responses of redox-active metalloenzymes immobilized on the electrode surface. − These techniques revealed that the four iron cofactors are sequentially reduced and the four-electron-reduced NOR is the active form for the NO reduction. , However, the assignments of previously reported redox potentials were unclear owing to the lack of monitoring specific molecular signatures during the redox measurements. Because vibrational spectroscopy is useful for characterization of the active site structure, vibrational spectroscopic measurements coupled with the PFE would be ideal to understand accurate redox behavior of metalloenzymes at the electrode interface. − …”

Surface Enhanced Infrared Characterization Using Disordered Slit-Antenna Arrays for the Detection of Electrodeposited Cytochrome C

“…It was recently found that the measured average SEIRA signal of slits is higher compared to their inverse rod structures for the same vibrational layer, showing their advantages for SEIRA experiments over the better-known solid nanoantennas [7,8]. Moreover, slits present yet another advantage with respect to rods which makes them best suited for the present work: the gold surface surrounding the slits remains conducting and allows combining SEIRA with electrochemical measurements, which is of great interest to study redox proteins [9,10].…”

Electrochemical Biosensors Based on Membrane-Bound Enzymes in Biomimetic Configurations

“…It was found that changing the way of constructing the system affected the amide I/amide II infrared bands intensity ratio, which might indicate different orientations or arrangements of the enzyme on the electrode. The SEIRA experiments showed that CpI was preferably incorporated into the fBLM with the catalytic hydrophilic arm towards the solution, which made it catalytically active on NADH oxidation and translocation of protons, thus acidifying the electrode/fBLM interface [82].…”

“…Although sBLMs and tBLMs have shown to be very valuable as model systems for fundamental studies of cell membrane processes [9,25,29,45,48], we think that fBLMs are more adequate for biosensor development. The presence of a water cushion in the interface between the fBLM and the electrode facilitates the insertion of transmembrane proteins in the lipid bilayer and gives higher flexibility and mobility within the biomimetic construction [77,78,82]. Moreover, the presence of an aqueous interphase facilitates charge transfer process across the membrane, which can help in coupling the biological recognition event to the electrochemical transductor [77,78].…”

“…Several surface characterization techniques are now available for electrochemical research, such as AFM/STM, SERS, SEIRAS, and PM-IRRAS, which allow for the complete characterization of the biomimetic constructions and immobilized membrane enzymes on the electrode [8]. The use of these surface characterization techniques combined with the diverse panoply of existing electrochemical methods permits correlating the configuration of the immobilized membrane enzyme in the biomimetic construction with its electrocatalytical properties [7,69,72,82]. In our opinion, this kind of studies should, in the future, considerably aid the optimization of biosensors design based on biomimetic systems.…”