Redox enzymes in tethered membranes.
An electrode surface is presented that enables the characterization of redox-active membrane enzymes in a native-like environment. An ubiquinol oxidase from Escherichia coli, cytochrome bo(3) (cbo(3)), has been co-immobilized into tethered bilayer lipid membranes (tBLMs). The tBLM is formed on gold surfaces functionalized with cholesterol tethers which insert into the lower leaflet of the membrane. The planar membrane architecture is formed by self-assembly of proteoliposomes, and its structure is characterized by surface plasmon resonance (SPR), electrochemical impedance spectroscopy (EIS), and tapping-mode atomic force microscopy (TM-AFM). The functionality of cbo(3) is investigated by cyclic voltammetry (CV) and is confirmed by the catalytic reduction of oxygen. Interfacial electron transfer to cbo(3) is mediated by the membrane-localized ubiquinol-8, the physiological electron donor of cbo(3). Enzyme coverages observed with TM-AFM and CV coincide (2-8.5 fmol.cm(-)(2)), indicating that most-if not all-cbo(3) on the surface is catalytically active and thus retains its integrity during immobilization.
Jeuken
Lars J C
LJ
Institute of Molecular Biophysics, School of Physics and Astronomy, Centre for Self-Organising Molecular Systems, and Astbury Centre for Structural and Molecular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom. L.J.C.Jeuken@leeds.ac.uk
Connell
Simon D
SD
Henderson
Peter J F
PJ
Gennis
Robert B
RB
Evans
Stephen D
SD
Bushby
Richard J
RJ
D002784:Cholesterol / Q000737:chemistry; D003573:Cytochrome b Group; D003580:Cytochromes / Q000737:chemistry* / Q000378:metabolism; D004563:Electrochemistry; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism; D029968:Escherichia coli Proteins; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism; D018625:Microscopy, Atomic Force; D008958:Models, Molecular; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism; D011510:Proteolipids / Q000737:chemistry / Q000378:metabolism; D020349:Surface Plasmon Resonance; D013499:Surface Properties
D003573:Cytochrome b Group; D003580:Cytochromes; D004800:Enzymes, Immobilized; D029968:Escherichia coli Proteins; D008051:Lipid Bilayers; D011510:Proteolipids; C083850:cytochrome bo3, E coli; C020485:proteoliposomes; D002784:Cholesterol; D010088:Oxidoreductases; C432586:ubiquinol oxidase