Orientational control on the physiological reaction of cytochrome c oxidase tethered to a gold electrode

Ataka, K.; Richter, B.; Heberle, J.

doi:10.1021/jp0534131

Journal Article

PreJuSER-51339

Orientational control on the physiological reaction of cytochrome c oxidase tethered to a gold electrode

Ataka, K. ; Richter, B. ; Heberle, J.FZJ*

2006
Soc. Washington, DC

The journal of physical chemistry <Washington, DC> / B 110, 9339 - 9347 (2006) [10.1021/jp0534131]

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Please use a persistent id in citations: http://hdl.handle.net/2128/715 doi:10.1021/jp0534131

Abstract: The physiological reaction of a membrane protein is reconstituted on a solid-supported electrode by orientational control via the position of an affinity tag. Recombinant cytochrome c oxidase (CcO) from Rhodobacter sphaeroides is immobilized on a chemically modified gold surface via the affinity of a histidine tag (His-tag) to a nickel chelating nitrilotriacetic acid surface. Control of the orientation is achieved by the adsorption of CcO through the His-tag engineered into the two opposite sites of the membrane protein surface. After reconstitution into a lipid layer, the functionality of this enzyme film electrode is probed by surface-enhanced infrared absorption spectroscopy and cyclic voltammetry. We demonstrate that cytochrome c (Cc) binds and initiates the catalytic reaction of CcO only when the latter is orientated with subunit II facing the bulk aqueous phase while Cc does not interact with the oppositely orientated CcO. We infer from the observed catalytic dioxygen reduction at potentials below 240 mV (vs a normal hydrogen electrode) that reduced Cc mediates electron input into CcO in a way similar to the physiological pathway. The quantitative analysis of the IR spectra indicates the presence of an inactive population of Cc bound to CcO at equal amounts as the redox-active population. This methodological approach demonstrates that the orientation of the membrane protein can be controlled depending on the position of the affinity tag. The approach is considered to be of general applicability as the introduction of affinity tags is routine in current biochemistry.

Keyword(s): Adsorption (MeSH) ; Electrochemistry (MeSH) ; Electrodes (MeSH) ; Electron Transport Complex IV: chemistry (MeSH) ; Electron Transport Complex IV: isolation & purification (MeSH) ; Electron Transport Complex IV: physiology (MeSH) ; Enzymes, Immobilized: chemistry (MeSH) ; Gold: chemistry (MeSH) ; Kinetics (MeSH) ; Membranes, Artificial (MeSH) ; Models, Molecular (MeSH) ; Recombinant Proteins: chemistry (MeSH) ; Rhodobacter sphaeroides: chemistry (MeSH) ; Spectroscopy, Fourier Transform Infrared (MeSH) ; Surface Properties (MeSH) ; Enzymes, Immobilized ; Membranes, Artificial ; Recombinant Proteins ; Gold ; Electron Transport Complex IV ; J