Monitoring the Transmembrane Proton Gradient Generated by Cytochrome bo3 in Tethered Bilayer Lipid Membranes Using SEIRA Spectroscopy.

ABSTRACT

Membrane proteins act as biocatalysts or ion/proton pumps to convert and store energy from ubiquitous environmental sources. Interfacing these proteins to electrodes allows utilizing the energy for enzymatic biofuel cells or other auspicious biotechnological applications. To optimize the efficiency of these devices, appropriate membrane models are required that ensure structural and functional integrity of the embedded enzymes and provide structural insight. We present a spectroelectrochemical surface-enhanced infrared absorption (SEIRA) and electrical impedance spectroscopy (EIS) study of the bacterial respiratory ubiquinol/cytochrome bo3 (cyt bo3) couple incorporated into a tethered bilayer lipid membrane (tBLM). Here, we employed a new lipid tether (WK3SH, dihydrocholesteryl (2-(2-(2-ethoxy)ethoxy)ethanethiol), which was synthesized using a three-step procedure with very good yield and allowed measuring IR spectra without significant spectral interference of the tBLM. The functional integrity of the incorporated cyt bo3 was demonstrated by monitoring the enzymatic O2 reduction current and the formation of the transmembrane proton gradient. Based on a SEIRA-spectroscopic redox titration, a shift of the pH-dependent redox potential of the ubiquinones under turnover conditions was correlated with an alkalinization of the submembrane reservoir by +0.8 pH units. This study demonstrates the high potential of tBLMs and the SEIRA spectroscopic approach to study bioenergetic processes.