Redox enzymes in tethered membranes

Area of Science:

• Biophysical Chemistry
• Electrochemistry
• Membrane Protein Biochemistry

Background:

• Characterizing membrane enzymes in native-like environments is crucial for understanding their function.
• Tethered bilayer lipid membranes (tBLMs) offer a platform for studying membrane proteins.
• Escherichia coli ubiquinol oxidase (cytochrome bo(3), cbo(3)) is a key respiratory enzyme.

Purpose of the Study:

• To develop and characterize an electrode surface enabling the study of redox-active membrane enzymes in a native-like environment.
• To investigate the functional integrity and catalytic activity of immobilized ubiquinol oxidase (cbo(3)).

Main Methods:

• Co-immobilization of ubiquinol oxidase (cbo(3)) into tethered bilayer lipid membranes (tBLMs) on functionalized gold surfaces.
• Structural characterization using surface plasmon resonance (SPR), electrochemical impedance spectroscopy (EIS), and tapping-mode atomic force microscopy (TM-AFM).
• Functional assessment via cyclic voltammetry (CV) and catalytic oxygen reduction assays.

Main Results:

• Successful formation and characterization of planar tBLMs incorporating cbo(3).
• Demonstrated catalytic activity of immobilized cbo(3) through oxygen reduction.
• Confirmed interfacial electron transfer mediated by ubiquinol-8.
• Coincident enzyme coverages from TM-AFM and CV indicate high catalytic activity and integrity.

Conclusions:

• The developed tBLM electrode surface provides a native-like environment for characterizing membrane enzymes.
• Immobilized cbo(3) retains its catalytic activity and structural integrity.
• This platform is suitable for studying electron transfer mechanisms in membrane-bound enzymes.