TY  - JOUR
AU  - Ataka, K.
AU  - Richter, B.
AU  - Heberle, J.
TI  - Orientational control on the physiological reaction of cytochrome c oxidase tethered to a gold electrode
JO  - The journal of physical chemistry  / B
VL  - 110
SN  - 1520-6106
CY  - Washington, DC
PB  - Soc.
M1  - PreJuSER-51339
SP  - 9339 - 9347
PY  - 2006
N1  - Record converted from VDB: 12.11.2012
AB  - 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.
KW  - Adsorption
KW  - Electrochemistry
KW  - Electrodes
KW  - Electron Transport Complex IV: chemistry
KW  - Electron Transport Complex IV: isolation & purification
KW  - Electron Transport Complex IV: physiology
KW  - Enzymes, Immobilized: chemistry
KW  - Gold: chemistry
KW  - Kinetics
KW  - Membranes, Artificial
KW  - Models, Molecular
KW  - Recombinant Proteins: chemistry
KW  - Rhodobacter sphaeroides: chemistry
KW  - Spectroscopy, Fourier Transform Infrared
KW  - Surface Properties
KW  - Enzymes, Immobilized (NLM Chemicals)
KW  - Membranes, Artificial (NLM Chemicals)
KW  - Recombinant Proteins (NLM Chemicals)
KW  - Gold (NLM Chemicals)
KW  - Electron Transport Complex IV (NLM Chemicals)
KW  - J (WoSType)
LB  - PUB:(DE-HGF)16
C6  - pmid:16671753
UR  - <Go to ISI:>//WOS:000237451300063
DO  - DOI:10.1021/jp0534131
UR  - https://juser.fz-juelich.de/record/51339
ER  -