Electron transfer processes between proteins or within complexes of biomolecules represent an important type of biochemical reactions and signal transfer. The temporary or permanent assembly of proteins within complexes guarantees the accessibility of reaction sites, short electron transfer distances and minimal interference by other species. Artificial assemblies with a defined signal transfer which mimic the biological example may have application potential in sensors but also in biofuel cell systems. One important issue is here the coupling of the biochemical reactions to the electrode surface. For several redox proteins electrode surfaces have been optimized in order to achieve efficient heterogeneous electron transfer, such as heme, iron-sulfur and copper proteins ^1-4.

Significant progress has been made in comparison to monolayer arrangements by use of the layer-by-layer-technique and the layered deposition of proteins on electrodes. Although the amount of protein can be significantly increased only the layers near to the surface are often able to exchange electrons directly with the electrode.

The presentation will give an overview on different approaches for the construction of fully electro-active protein multilayers. The assembly is based on the layer-by-layer deposition of the redox protein cytochrome c and a negatively charged second building block on electrodes, which can be the polyelectrolyte sulfonated polyanilline, carboxy-terminated gold or silica nanoparticles or a natural polymer dsDNA^5-8. The unique property of these systems is the defined increase of electro-active protein amount with the number of deposited layers. The increase in electro-active amount can be advantageously used also for the detection of superoxide radicals with enhanced sensitivity⁶. The different building blocks however will influence the properties of the system. The succesfull assembly process is a first precondition for a functioning system and has been analysed by SPR or QCM, showing different behavior for dsDNA based systems compared to the particles and polyelectrolyte based assemblies.

From the mechanistic point of view it is obvious that not only a single system can be used for the construction of electro-active protein multilayers, however the question remains how the electrons are transferred through the layered system towards the electrode. Different arguments have been collected in different studies that electron exchange between neighboring cyt c molecules might be the dominating mechanism^9-11. However, mainly building blocks have been used which posses conducting properties by themselfs under certain circumstances. Recently silica nanoparticles have been prepared, modified with carboxylic groups and applied as building blocks for such protein assemblies. Since also here efficient electron transfer through several protein layers has been found, the model of a electron hopping between the immobilized cyt c molecules can be strongly supported. However, a flexibility at least on the rotational level, seems to be essential for the functioning of the system since covalent crosslinking of the cyt c molecules results in a loss of electroactivity.

These layered assemblies of a redox protein can be combined with enzymes in order to establish signal chains with multiple step electron transfer reactions following natural examples. Thus sensing electrodes for the enzyme substrate can be constructed. Signal transfer can be achieved by an internally generated shuttle molecule as shown for xanthin oxidase – cyt c layers¹² or more advantageously by direct protein-protein electron transfer without the need of any internal or external shuttle molecule. This can be shown for billirubin oxidase (BOD), laccase, sulfite oxidase and more recently cellobiose dehydrogenase (CDH) ^13-16.

For example BOD can be co-immobilised with cyt c in multiple layers by means of a polyelectrolyte. The enzyme is catalytically active for oxygen reduction. The interesting properties are here that both reaction partners are immobilized on the electrode and that the catalytic oxygen current is increasing with the number of deposited layers. This means that BOD molecules immobilized in the outer layers still can communicate with the electrode by electron transfer from neighboring cyt c molecules. It has to be mentioned here that a rather high excess of cyt c has to be used during the assembly in order to connect the BOD molecules and transfer the electrons through the structure; higher amounts of enzyme disrupt the electron transport pathways ¹³. Studies with mutant forms of cyt c demonstrate that a smaller self exchange rate lower the efficiency of electron transport through the system. However, besides the self exchange also the assembly properties and the reaction rate with the enzyme are determining factors¹⁴.

The electron transfer can also occur in opposite direction as shown for assemblies with embedded CDH. Here electrons delivered by the oxidation of cellobiose or lactose have to be transported towards the electrode. Investigations of this bi-protein system also show that the interprotein electron transfer can be improved when the enzyme is used in a deglycosylated form. Much higher catalytic currents have been found.

References

1. M. J. Eddowes and H. A. O. Hill, Journal of the Chemical Society-Chemical Communications, 771-772 (1977)
2. S. Song, R. A. Clark, E. F. Bowden and M. J. Tarlov, Journal of Physical Chemistry, 97, 6564-6572 (1993).
3. K. A. Vincent and F. A. Armstrong, Inorganic Chemistry (2005) 44, 798-809.
4. O. Ikeda, M. Ohtani, T. Yamaguchi and A. Komura, Electrochimica Acta, 43, 833-839 (1998).
5. M. K. Beissenhirtz, F. W. Scheller, W. F. M. Stocklein, D. G. Kurth, H. Möhwald and F. Lisdat, Angewandte Chemie, 43, 4357-4360 (2004).
6. M. K. Beissenhirtz, F. W. Scheller and F. Lisdat, Analytical Chemistry, 76, 4665-4671 (2004).
7. S. Bonk, F. Lisdat, Biosensors and Bioelectronics, 25 (4), 739-744 (2009).
8. D. Sarauli, J. Tanne, D. Schäfer, I. W. Schubart, F. Lisdat, Electrochemistry Communications, 11 (12), 2288-2291 (2009).
9. J. Grochol, R. Dronov, F. Lisdat, P. Hildebrandt and D. H. Murgida, Langmuir, 23, 11289-11294 (2007).
10. R. Dronov, D. G. Kurth, H. Möhwald, F. Scheller, J. Friedmann, D. Pum, U. B. Sleytr and F. Lisdat, Langmuir, 24 (16), 8779-8784 (2008).  
11.  F. Lisdat, R. Dronov, H. Möhwald, F.W. Scheller, D.G. Kurth, Chemical Communications, 3, 274-283 (2009).
12. R. Dronov, D. G. Kurth, H. Möhwald, F. W. Scheller and F. Lisdat, Electrochimica Acta, 53, 1107-1113 (2007).
13. R. Dronov, D. G. Kurth, H. Möhwald, F. Scheller and F. Lisdat, Angewandte Chemie, 47, 3000-3003 (2008).
14. F. Wegerich, P. Turano, M. Allegrozzi, H. Möhwald, F. Lisdat, Langmuir, 27 (7), 4202-4211 (2011).
15. T. Balkenhohl, S. Adelt, R. Dronov and F. Lisdat, Electrochemistry Communications (2008) 10, 914-917.
16. R. Dronov, D. G. Kurth, H. Möhwald, R. Spricigo, S. Leimkuehler, U. Wollenberger, K. V. Rajagopalan, F. W. Scheller and F. Lisdat, Journal of the American Chemical Society, 130, 1122 (2008).

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1. Coupling of pyrroloquinoline quinone dependent glucose dehydrogenase (PQQ-GDH) with CytC/DNA multilayer electrodes
2. Electrochemical observation of the thermally induced phase transition for a switchable polymer immobilised onto gold electrodes