Characterization of cytochrome bo3 activity in a native-like surface-tethered membrane

Abstract

We have developed a simple native-like surface-tethered membrane system to investigate the activity of cbo(3) (cytochrome bo(3)), a terminal oxidase in Escherichia coli. The tethered membranes consist of E. coli inner-membrane extracts mixed with additional E. coli lipids containing various amounts of the cbo(3) substrate UQ-10 (ubiquinol-10). Tethered membranes are formed by self-assembly from vesicles on to gold electrodes functionalized with cholesterol derivatives. cbo(3) activity was monitored using CV (cyclic voltammetry) with electron transfer to cbo(3) mediated by UQ-10. The apparent K(m) for oxygen with this system is 1.1+/-0.4 microM, in good agreement with values reported in the literature for whole-cell experiments and for purified cbo(3). Increasing the concentration of lipophilic UQ-10 in the membrane leads to an increase in cbo(3) activity. The activity of cbo(3) with long-chain ubiquinones appears to be different from previous reports using short-chain substrate analogues such as UQ-1 in that typical Michaelis-Menten kinetics are not observed using UQ-10. This native-like membrane model thus provides new insights into the interaction of transmembrane enzymes with hydrophobic substrates which contrasts with studies using hydrophilic UQ analogues.

Model Membrane Platforms for Biomedicine: Case Study on Antiviral Drug Development

“…This flexibility enables the determination of binding saturation values that are more representative of biological systems. In addition to model membranes, sensing platforms can consist of cell-derived membranes that contain a natural assortment of components including lipids, proteins, and glycosaminoglycans [77][78][79]. While model membranes represent a general platform to study membrane association processes driven by amphiphilic interactions, cell-derived membranes contain receptors which can mediate specific binding of proteins.…”

Extracellular Electron Transport-Mediated Fe(III) Reduction by a Community of Alkaliphilic Bacteria That Use Flavins as Electron Shuttles

“…Ultraflat template-stripped gold (TSG) electrodes (surface area [A] ϭ 0.2 cm 2 ) were prepared and cleaned (see reference 58 for details). Self-assembled monolayers (SAMs) were formed on electrodes by incubating them with 1 mM 8-mercaptooctanol in propanol for 16 h. After rinsing with propanol and methanol, the electrodes were dried under a nitrogen gas flow and assembled in a bespoke glass electrochemical cell (58). Voltammetry was conducted with a standard 3-electrode setup.…”

Photosynthetic Proteins in Supported Lipid Bilayers: Towards a Biokleptic Approach for Energy Capture

“…The data suggested the existence of both electrostatic and amino group specific interactions between cytochrome c and CX, which opens the field to the design and production of surface based biosensors. [32e] Other studies have used different approaches, such as cytochrome sensing based on the reaction between cytochrome c and cytochrome c oxidase, the activity of the cytochrome as a function of ubiquinol‐10 in SLBs based on cyclic voltammetry ( Figure ), and the effect of Zn ions on the proton release of cytochrome . Although there are some reports about proton pumping by cytochrome c reconstituted within vesicles, there is still less progress in these experiments carried out on SLBs.…”