Elsevier

Electrochimica Acta

Volume 52, Issue 25, September 2007, Pages 7307-7314
Electrochimica Acta

Key role of the anchoring PEI layer on the electrochemistry of redox proteins at carbon electrodes: Consequences on assemblies involving proteins and clay

https://doi.org/10.1016/j.electacta.2007.06.003Get rights and content

Abstract

The key role of positively charged anchoring films on the electroactivity of various bacterial proteins at a pyrolytic graphite electrode is discussed. Several iron-containing low redox potential proteins, either negatively charged or positively charged were considered. Using quartz crystal microgravimetry (QCM) and electrochemical techniques, it is demonstrated that electron transfer on these proteins can be inhibited or promoted essentially depending on the electrostatic interactions with the positive PEI layer. Consequences of this interaction on the electroactivity of proteins involved in assemblies with clays are discussed. In particular, although bacterial polyhemic cytochrome c3 is able to form stable LBL assemblies with intercalated clay layers, electroactivity of the so-mounted construction is highly dependant on the structure of the anchoring layer.

Introduction

Electron transfer between a protein and an electrode surface can be considered as a mimetic process of the electron transfer through redox molecules chains in biological systems. One way to be closer to in vivo conditions is to build up onto the electrode surface ultrathin enzymes or proteins layers, which are supposed to be organized in such a way that high enzymatic specificity and hence activity is reached. This can be achieved using layer-by-layer (LBL) self assembly of oppositely charged layers [1], a process which looks very much like the physiological recognition of redox partners before electron transfer, mainly based on electrostatic interactions.

In this way, negatively charged montmorillonite nanoparticles were alternatively stacked with redox proteins and enzymes, such as cytochromes, myoglobin, hemoglobin or glucose oxidase [2], [3], [4], [5], [6], [7]. Electroactivity of the redox molecules within the so-mounted architecture was demonstrated. We have shown in a previous work [8] that LBL assemblies involving clay and bacterial cytochrome c3 were possible and they allowed satisfactory catalytic efficiencies, especially toward H2 uptake/evolution reaction. This was however achieved using the property of self-adsorption of cytochrome c3 onto the pyrolytic graphite electrode as a first step of the LBL assembly. This procedure leads to a short life cycle assembly due to the progressive denaturation of the adsorbed cytochrome c3. Otherwise, direct adsorption of clay nanoparticles onto electrode surfaces leads to more or less instable layers. Thus, advantage can be gained due to the attachment of polymeric cations onto the electrodes. This first layer is expected to play the role of an anchoring layer, stabilizing the whole assembly. Actually, the use of montmorillonite (M) as polyanion in an alternate adsorption process with linear polymeric cations such as poly(ethylenimine) (PEI) was proposed a few years ago by Lvov et al. [1]. Polycation “glue” was also demonstrated to help in the assembly of negatively charged M and positively charged proteins [9].

On the other hand, in the peculiar case of biological materials, electron transfer rate between oxidoreduction partners is largely dependent on the formation of protein–protein complexes. Specific binding requirements involving hydrophobic, hydrogen-bonding but above all electrostatic interactions provide much of the specificity that is essential in biological electron transfer systems. Similarly, it is widely assumed that electrostatic environment is the most crucial parameter for direct electron transfer between a protein and an electrode to be achieved. Polycationic anchoring layers onto the electrode surface are thus expected to play a key role in the electron transfer process throughout assemblies alternating clays and proteins. Careful examination of the influence of the PEI film structure on the electroactivity of proteins is needed for efficient construction at an electrode surface.

In this work, after the structure of the PEI layer is discussed at a PG electrode, the role of the cationic anchoring layer on the electron transfer on redox proteins is studied. Redox proteins differing in their global charge, but having redox potentials in the same range, are considered: one non-hemic protein, spinach ferredoxin, and several bacterial multihemic cytochromes c3. Due to their potentialities as physiological partners of hydrogenases in biotechnological processes for hydrogen production/consumption, the study is focused on the behavior of cytochromes c3. The consequences of the anchoring PEI layer structure on the electroactivity of cytochromes c3 in alternate construction with clay are finally discussed.

Section snippets

Materials

Spinach ferredoxin was purchased from Sigma. Cytochromes c3 from Desulfovibrio vulgaris Hildenborough (DvH), Desulfomicrobium norvegicum (Dn) and Desulfovibrio africanus (Da) were prepared and purified as previously described [10], [11], [12]. The numbering of the hemes is the same as the numbering found in the cytochromes c3 structures in the PDB data files, and is made according to the rank of binding of the heme to the cysteine residues of the polypeptide chain. Poly(ethylenimine) (PEI, MW

Structural considerations on PEI adsorbed layer at the PG electrode

A preliminary CV study was realized using Fe(CN)63− and Ru(NH3)63+ as charged probes, with the main goal of examining structural aspects of adsorbed PEI onto the PG electrode (curves not shown). Modification of the PG surface by PEI adsorption resulted in an increase in the charge transfer rate for Fe(CN)63−, as denoting by the decrease of ΔEp from 170 mV at the bare electrode to 65 mV at the {PEI}-modified PG electrode. On the other hand, inhibition of the electron transfer on Ru(NH3)63+ was

Conclusion

Montmorillonite and cationic polyelectrolyte have the capability to form stable LBL assemblies. This procedure was considered as a promising tool to embody proteins in LBL architecture. However, we have demonstrated in this work that the first anchoring layer onto the electrode is the crucial point for the electron transfer onto the protein to be achieved. The electrochemical behavior of different proteins either in solution or embedded in assemblies with clays well illustrates this assumption.

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