Book of Abstracts - Ruhr-Universität Bochum

P-58
ISBOMC `10 5.7 – 9.7. 2010 Ruhr-Universität Bochum
Affinity Binding for Controlled Orientation and Electrochemistry in Self
Assembled Monolayers of Reductases.
Nicolas Plumeré, a Ellen R. Campbell, b Bill H. Campbell b
a Ruhr Universität Bochum, Center for Electrochemical Science, Universitätsstrasse 150, 44780,
Bochum, Germany. b NECi, 334 Hecla Street, Lake Linden, USA. E-mail: nicolas.plumere@rub.de
Metal-chelating ligands have been designed in the field of immobilized metal ion affinity
chromatography 1 (IMAC) for the purification of polyhistidine-tagged (His-tagged) proteins. Dense
monolayers of similar metal-chelating ligands on electrode surfaces have recently been applied for the
immobilization and controlled orientation of His-tagged proteins via affinity binding. 2–4 In particular,
nitrilotriacetic (NTA) and imminodiacetic (IDA) acid terminated gold and carbon electrodes were used
for the immobilization of laccase, 4 glucose oxidase, 5 horseradisch peroxidase 2 and hemoprotein. 3 In
these cases, the attachment of the enzyme via His-tag yields fully active protein films in the presence
of electron mediators. In some cases, direct electron transfer was observed as well. 5 Cu 2+ and Ni 2+
were used as the metal cations.
However, the use of affinity binding involving Ni 2+ or Cu 2+ is limited to applications that require redox
potentials less negative than the reduction potential of the metal cation complexes. 2 Therefore, most
reductases cannot be used in such systems.
In order to overcome this limitation, we used Zn 2+ as the metal cation for the coordination of a Histagged
reductase. Nitrate reductase (NaR) as the enzyme and methyl viologen as the electron mediator
were chosen to test the affinity binding via Zn 2+ cations on NTA modified glassy carbon electrodes.
The electrochemical investigations of the NaR monolayer on NTA-Zn 2+ films demonstrate the activity
for the catalytic reduction of nitrate to nitrite in presence of methyl viologen. The catalytic current
density corresponds to the one expected for a fully active enzyme monolayer. Moreover, the reduction
of the Zn 2+ is not observed at the potential necessary for the reduction of methyl viologen. Therefore,
affinity binding based on Zn 2+ may be used for the immobilization of Nitrate reductases in their active
form.
References
1. G. S. Chaga J. Biochem. Biophys. Methods 2001, 49, 313-334.
2. R. Blankespoor, B. Limoges, B. Schöllhorn, J.-L. Syssa-Magalé, D. Yazidi Langmuir 2005, 21,
3362-3375.
3. V. Balland, S. Lecomte, B. Limoges Langmuir 2009, 25, 6532-6542.
4. V. Balland, C. Hureau, A. M. Cusano, Y. Liu, T. Tron, B. Limoges Chem. Eur. J. 2008, 14, 7186-
7192.
5. S. Demin, E. A. H. Hall, Bioelectrochemistry 2009, 76, 19-27.
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