Interaction of Flavin-Dependent Fructose Dehydrogenase with

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Interaction of Flavin Dependent Fructose Dehydrogenase with Cytochrome c as Basis for the Construction of Biomacro-molecular Architectures on Electrodes Christoph Wettstein, Kenji Kano, Daniel Schäfer, Ulla Wollenberger, and Fred Lisdat Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b00815 • Publication Date (Web): 23 May 2016 Downloaded from http://pubs.acs.org on June 4, 2016

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Analytical Chemistry

Interaction of Flavin-dependent Fructose Dehydrogenase with Cytochrome c as Basis for the Construction of Biomacromolecular Architectures on Electrodes Christoph Wettstein‡, Kenji Kano†, Daniel Schäfer‡, Ulla Wollenberger§, Fred Lisdat*,‡ ‡

Biosystems Technology, Institute of Applied Life Sciences, Technical University of Applied Sciences Wildau, Hochschulring 1, 15745 Wildau, Germany † Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo, Kyoto 606–8502, Japan § Institute of Biochemistry and Biology, University Potsdam, Karl-Liebknecht-Str. 24-25, 14476 Potsdam/ Golm, Germany ABSTRACT: The creation of electron transfer (ET) chains based on the defined arrangement of enzymes and redox proteins on electrode surfaces represents an interesting approach within the field of bio-electrocatalysis. In this study, we investigated the ET reaction of the flavin-dependent enzyme fructose dehydrogenase (FDH) with the redox protein cytochrome c (cyt c). Two different pH optima were found for the reaction in acidic and neutral solutions. As cyt c was adsorbed on an electrode surface while the enzyme remained in solution, ET proceeded efficiently in media of neutral pH. Inter-protein ET was also observed in acidic media; however, it appeared to be less efficiently. These findings suggest that two different ET pathways between the enzyme and cyt c may occur. Moreover, cyt c and FDH were immobilized in multiple layers on an electrode surface by means of another biomacromolecule: DNA (double stranded) using the layer-by-layer technique. The bi-protein multilayer architecture showed a catalytic response in dependence on the fructose concentration, indicating that the ET reaction between both proteins is feasible even in the immobilized state. The electrode showed a defined response to fructose and a good storage stability. Our results contribute to the better understanding of the ET reaction between FDH and cyt c and provide the basis for the creation of all-biomolecule based fructose sensors the sensitivity of which can be controlled by the layer preparation.

genase (GDH).16 These protein assemblies possess the potential of application in the field of bioelectronics, biosensors and biofuel cells.8,17,18 In this study, a membrane-associated enzyme fructose dehydrogenase (FDH) was applied. FDH was purified to homogeneity and characterized by Ameyama et al.19 for the first time. FDH is a hemoflavo-protein with a molecular mass of approx. 140 kDa, consisting of three subunits (67.0 kDa, 50.8 kDa, 19.7 kDa). The 67.0 kDa subunit holds a flavin-adeninedinucleotide (FAD) and the 50.8 kDa subunit has three hemes c as prosthetic groups, whilst the smaller one is also responsible for membrane anchoring.20, 21 The ET pathway in direct electrochemical communication with electrodes has been investigated, revealing that ET proceeds via heme C3 and C2, by doing C1 remains reduced. The formal redox potential (Ef) of the three heme sites was determined to be -10±4 mV, 60±8 mV and 150±4 mV vs. Ag/AgCl (sat. KCl) at pH 5.0.21 Comparing these values with other studies shows that the Ef of C2 (i.e. 60±8 mV) is in the range of the Ef determined for FDH adsorbed on gold22 and on carbon black23 with an Ef of +80 mV and +39 mV vs. Ag/AgCl (sat. KCl), respectively. The Km value of the enzyme for fructose was determined to be 10 mM at pH 4.5 with K3[Fe(CN)6] as electron acceptor.19 FDH is an acidic enzyme with an pI of 5.0±0.1, its pH opti-

INTRODUCTION Electron transfer (ET) reactions play a crucial role in the metabolic pathways of all organisms. They occur in essential biochemical cascades, such as the respiratory chain and photosynthesis and are enabled by precisely organized proteins and protein complexes.1 Based upon the natural example, several protein assemblies have been engineered, mimicking ET cascades of biochemical signal transduction. Acting as an electron shuttle in the respiratory chain, cyt c has been studied extensively by electro-analytical techniques. The protein shows direct electron transfer (DET) on modified electrode surfaces, such as self-assembled monolayer (SAM)-covered gold.2-5 Here, the use of electron mediators is avoided, to reduce potential interferences and side reactions. In addition, cyt c can be arranged in multiple layers on electrodes and is able to transfer electrons by self-exchange.6-8 Functioning as recognition elements of specific substrates, enzymes can be linked via cyt c to different electrodes. Thus, bioelectrochemical signals are generated in the presence of the substrate and can be recorded by different voltammetric methods. Such reactions do not only occur with one immobilized protein,9-11 but can also be extended to the fixed state of both reaction partners.8,12 This has been shown for the molybdo-enzyme sulfite oxidase,13,14 the flavo-enzyme cellobiose dehydrogenase (CDH)15 and the PQQ-dependent glucose dehydro-

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mum is at 4.0 to 4.5 and its temperature optimum at 37 °C. Due to the hydrophobic surface patches, FDH tends to precipitate in solution, leading to inactivation. However storage of the enzyme in Triton X®-containing buffer at 4 °C preserves the activity for at least two weeks.19 Instead of FAD pyrroloquinoline quinone (PQQ) was also described as a cofactor of FDH24-26. However, to the best of our knowledge no studies addressing the identification of PQQ as the enzymes cofactor are existent. Therefore, all the references consulted here go back to the studies of Ameyama et al.,19 who indicated FAD as the cofactor. The first direct evidence of FAD was given by a report by Kawai et al..20 In recent biotechnological studies, FDH was used for the construction of biosensors27-29 and biofuel cells.23,30,31 Its immobilization was shown to be feasible and ET was found to proceed either directly or via mediating molecules. Carbonbased materials were found to be a suitable interface to achieve direct ET (DET).23,32,33 Based upon the potential at which DET is observed, it seems that the orientation of the enzyme with the heme site facing the surface facilitates an effective ET between FDH and the electrode. Tominaga et al.32 generated biolectrocatalytic currents on basal-plane, highly oriented pyrolytic graphite and plastic formed carbon plate. The catalytic oxidation currents were only observed in acidic solutions with a pH ≤ pH 6.0. In neutral and alkaline media, no catalytic reaction was observed, however, evidence for the decomposition of the FDH trimer was found here. The most efficient system using a carbon based electrode has been established on Ketjen black23 achieving high current densities at pH 5.0. Besides carbon, gold was found to be a favourable surface for ET with FDH. For example, the deposition of citrate-reduced gold nanoparticles modified with mercaptoethanol resulted in a bioelectrocatalytic response in the presence of fructose.34 Furthermore, polyaniline modified ITO electrodes have been shown to allow efficient DET.35 Ferapontova et al.36 showed DET at cysteamin- and mercaptoundecanol (MU)-modified gold electrodes, with FDH in solution. Cyt c was described as mediating protein for electrons towards the MU-modified electrode while this reaction was found to be most efficient at pH 7.0. Within this study, the inter-protein ET between FDH and cyt c has been characterized in solution and in a surface confined state. Targeting the creation of an ET chain for analytical purpose, the immobilization of both components on the electrode surface has been investigated. Consequently, a fructose sensitive electrode with tunable sensitivity has been obtained.

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trifugal filters with a MWCO of 10 kDa were provided by Merck Millipore Ltd. (Cork, Ireland). All solutions were prepared in 18 MΩ Millipore-water (Eschborn, Germany). Commercially available FDH from Gluconobacter japonicus NBRCA3260 (formerly Gluconobacter industrius)20 was provided by Sigma Aldrich as lyophilized powder additionally containing salts and agents for stabilisation, i.e. detergents, antioxidants and sugars, to prevent inactivation. According to the provider the purchased sample of 4.5 mg FDH contained 5.1 % protein. A 0.5 mg (protein)/ml stock (i.e. 3.3 µM) solution was prepared after dissolving the lyophilisate in 0.5 ml McIlvaine buffer (pH 4.5). Alternatively FDH from Gluconobacter japonicus NBRCA3260 was provided by the working group of Prof. Dr. Kenji Kano (Kyoto University, Japan). The enzyme was recombinantly expressed in Gluconobacter oxydans and purified as described by Kawai et al.20 It was dissolved in a 5.6 mg/ml stock solution (100 mM McIlvaine pH 6.0), containing 300 mM sucrose for stabilization. Its specific activity was determined prior to use as described previously.19 The activity test is based on ferricyanide reduction by FDH in the presence of fructose. The reaction was stopped by the addition of phosphoric acid, SDS and iron(III) sulfate containing solution. SDS denatures the enzyme and thus disables further reduction of ferricyanide. Iron(III) sulfate reacts with the reaction product (ferrocyanide) to Prussian blue, which is detected spectrophotometrically at 660 nm. UV-Vis spectroscopy. Kinetic measurements were performed with a Thermo Scientific Evolution 300 spectrometer (Weltham, MA, USA) equipped with a Peltier element for temperature control. As control 1 Unit (U) of FDH was added to a 25 µM cyt c solution in 100 mM McIlvainebuffer at different pH values. Each sample was mixed by 5 s stirring and equilibrated to 37 °C for 5 min, prior to measuring. Subsequently, fructose was added to the cyt c/FDH mixture to obtain a concentration of 50 mM, the solution was stirred again for 5 s and equilibrated to 37 °C. Now the reduction rate of cyt c was followed at 550 nm for 5 min. For the calculation of the initial rate of cyt c reduction, the velocity (ν) was calculated as the slope of the kinetic curve in the linear phase of the cyt c reduction after subtraction of the substrate free control. By applying the law of Lambert-Beer, using an extinction coefficient (ε) of reduced cyt c (with ε(550)red – ε(550)ox = 21.1 mM-1cm-1),42 the extinction recorded at 550 nm and the thickness of the irradiated body (i.e. 1 cm), the converted substrate concentration was determined. As a control, the absorbance of fully reduced cyt c was determined additionally by adding ascorbic acid to the pH 4.5 sample. Preparation of protein electrodes. Gold wire electrodes (AuE) were cleaned by three times incubating in piranha solution (H2SO4/H2O2, 3:1) for 20 min. After each step, the electrodes were sonicated in the same solution and thoroughly rinsed with ultrapure water. In the next step, the electrodes were incubated for 4 h in boiling 2.5 M KOH, rinsed again in ultrapure water and stored in 96 % H2SO4. Prior to modification with the MU:MUA SAM, the AuEs were incubated for 20 min in 65 % HNO3, rinsed with ultrapure water, then with EtOH and dried. The so prepared AuEs were incubated in 5 mM ethanolic solution of MU:MUA

EXPERIMENTAL SECTION Chemicals. Citric acid, D-fructose, iron(III) sulfate, K3[Fe(CN)6], KOH, 11-mercapto-1-undecanol (MU), 11mercaptoundecanoic acid (MUA), Triton X®-100 (TX), cytochrome c (cyt c) from horse heart and dsDNA from calf thymus were purchased from Sigma-Aldrich (Taufkirchen, Germany). Ethanol (EtOH), sodium dodecylsulfate (SDS), Na2HPO4, H2SO4 (96 %), H3PO4 (≥ 85 %), H2O2 (30 %) and HNO3 (≥ 65 %) were provided by Roth (Karlsruhe, Germany). KCl and K2HPO4/KH2PO4 (KPi buffer) were obtained from Merck. Gold wire (0.5 mm diameter) was obtained from Goodfellow (Bad Nauheim, Germany). Amicon Ultra cen-

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(3:1) for at least 24 h. MU and MUA form a self-assembling monolayer (SAM) on the electrode surface. The negatively charged carboxylic acid group of MUA attracts positively charged surface patches of cyt c. MU serves as a spacer between the MUA molecules to keep distance between the negatively charged carboxylic groups of MUA.4 Preparation of cyt c monolayer electrodes. Cyt c monolayers were prepared on the MU:MUA modified electrodes by running CV scans in a 30 µM cyt c solution (5 mM KPi pH 7.0) at a scan rate (SR) of 100 mV/s in a potential range of -0.4 V to 0.4 V until a stable signal was obtained. Preparation of cyt c monolayers with immobilized FDH. Cyt c monolayer electrodes were prepared as described above and incubated in a 200 µg/ml FDH solution with approximately 100 U/ml in 5 mM KPi buffer pH 7.0. The buffer exchange from 100 mM McIlvaine (pH 4.5) to 5 mM KPi buffer (pH 7.0) was performed by filling a 20 µl FDH stock solution up to 500 µl with cold KPi buffer (4 °C) and centrifuging for 9 min at 12000 rpm and 4 °C in a spin column with a MWCO of 10 kDa, resulting in a final volume of about 50 µl. Prior to measuring the cyt c-FDH electrode was rinsed by gently dipping 5x in buffer 5 mM KPi buffer pH 7.0. Preparation of cyt c:FDH multilayer electrodes. A mixture of 20 µM cyt c and 0.8 µM FDH was prepared in 0.5 mM KPi buffer pH 5.0. The ratio of cyt c and FDH was varied between 100:1 and 5:1, keeping a cyt c concentration of 20 µM. To remove detergent residues from the enzyme stock solution, which may disturb the multilayer assembly, the solution was washed three times with the same buffer, using Amicon Ultra centrifugal filters with a MWCO of 3 kDa (12000 rpm for 9 min at 4 °C). Directly after the preparation of the cyt c monolayer, the DNA/(cyt c:FDH) multilayer architecture was assembled on top of the monolayer electrode. Therefore, freshly prepared monolayer electrodes were incubated alternately in (a) 0.2 mg/ml DNA (0.5 mM KPi buffer pH 5.0) and (b) a mixture of cyt c and FDH for 10 min each. After each step, the electrode was washed by gently dipping it 5x in 0.5 mM KPi buffer (pH 5.0). To stabilise the multilayer assembly the electrodes were dried at room temperature (RT) over night; the next day the modified electrodes were incubated in 0.5 mM KPi buffer pH 5.0 at 40 °C for 40 min prior to use.44 Cyclic voltammetry. Cyclic voltammetry measurements were conducted with an Autolab PGSTAT 20 (Metrohm, Germany). The mono and multilayer electrodes (working electrode, WE) were placed in a custom-made 1 ml cell, immersion depth was 2 mm (surface area = 3.3 mm²). As reference electrode (RE) an Ag/AgCl (1 M KCl) electrode (Microelectrodes Inc., Bedford, USA) with a potential of +0.236 V vs. NHE43 and a platinum wire as counter electrode (CE) were used. After preparing the electrodes as described above, the surface coverage of electro-active cyt c was determined in 5 mM KPi pH 7.0 using a scan rate (SR) of 100 mV/s. Faraday’s law, which correlates the adsorbed mass to the transferred charge was applied, calculating the charge as the average value of the integrated area underneath the oxidation and the reduction peak. For the measurements, the WE was first equilibrated in the measuring buffer, i.e. 5 mM KPi buffer pH 7.0 or 20 mM KPi buffer pH 5.0 (and 4.5), for at least 2 min. After the multilayer was assembled and heat stabilised

in 0.5 mM KPi buffer pH 5.0 (see above) the electrodes were equilibrated in 5 mM KPi buffer pH 7.0 for 10 min. Prior to measuring, FDH or fructose, were added in different steps, and the buffer solution was gently stirred for 30 s. Catalytic currents were analysed at +200 mV vs. Ag/AgCl (1 M KCl) at a SR of 2 mV/s. For each measurement at least two cycles were run, starting from -150 mV vs. Ag/AgCl (1 M KCl). The second cycle is shown in the respective graphs. SPR measurements. SPR measurements were performed on a Biacore T100 (GE Healthcare) at a fixed flow rate of 1 µl/min with intermittent buffer flow and a temperature of 25 °C. Before use, the unmodified gold sensor chips (SIA KitAu, BT-1004-0, GE Healthcare) were cleaned twice in a 3:1 solution of H2SO4 (96 %) and H2O2 (30 %) for 10 min and rinsed with water and EtOH. The clean gold chips were modified with a MU:MUA SAM (48 h ethanolic 3:1 MU:MUA solution), washed with EtOH and dried at RT. Being ready to use, the so prepared chip was placed in the SPR device. In analogy to the gold electrode preparation, cyt c was first adsorbed on the SAM as a monolayer (5 mM KPi buffer pH 7.0). For the multilayer assembly, the buffer was changed to 0.5 mM KPi buffer pH 5.0 and the DNA solution as well as the cyt c:FDH mixture were flushed over the chip surface. RESULTS AND DISCUSSION Investigation of freely diffusing cyt c and FDH. The pHprofile of the ET reaction between FDH and cyt c was studied in the range of pH 4.5 to 7.0 by determining the velocity (v) of the cyt c reduction. After mixing cyt c and FDH, fructose was added and the absorbance change of the reduced cyt c was recorded at 550 nm. During the measurement, no saturation of absorbance was observed, verifying that the kinetic data were recorded in the linear range of cyt c reduction. Figure 1 shows that the reduction of cyt c by FDH proceeds most efficiently at pH 4.0 since here the highest rate was detected. The rate decreased with increasing pH values. However, when reaching pH 6.5 and 7.0 the value increased again.

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Figure 1. Velocity (v) of cyt c reduction in the presence of FDH and the substrate in solutions of different pH values (100 mM McIlvaine buffer, [cyt c] = 25 µM, FDH = 1 U/ml, [fructose] = 50 mM). The reaction rate was measured by following the cyt c reduction at 550 nm and calculated from the change in absorbance versus time using the Lambert-Beer equation. Here FDH was used provided by Sigma.

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This is in contrast to the behaviour of the enzyme when performing the activity assay using K3[FeCN]6 as an electron acceptor,19 since here a high enzyme activity was verified only in acidic media; at neutral pH only a minor activity remained (i.e. at pH 7.0 approx. 4 %). Since for the reaction of FDH with cyt c two pH optima exist, this may suggest different mechanisms of interaction: (i) ET may either proceed from the active site (FAD containing subunit) via the heme groups (subunit with three heme centers) towards cyt c or (ii) directly from the FAD-subunit to cyt c. A similar conclusion was drawn by Ferapontova et al.36 after investigating ET between cyt c and FDH by cyclic voltammetry at different scan rates (SRs). The surprisingly high activity of FDH interaction with cyt c in neutral solutions was also found in this study. It has to be added here, that electron transfer is downhill for both routes and recent findings indicate that the heme with the largest redox potential is not taking part in the ET chain during fructose conversion.21 Investigation of surface-bound cytochrome c in reaction with freely diffusing FDH. Neutral pH. For the investigation of the interaction between surface confined cyt c and FDH in solution, cyt c was adsorbed on a mercaptoundecanol/ mercaptoundecanoic acid (MU:MUA)-modified gold electrode as a monolayer. Here the fast and well-characterized heterogeneous electron transfer of adsorbed cyt c provides a profound basis for the interaction study with the enzyme.4 The cyt cFDH reaction was first studied at pH 7.0, since the pH profile of the interaction suggests an efficient enzymatic reduction of the redox protein at neutral pH. Figure 2A shows the cyclic voltammogram (CV) of adsorbed cyt c in the presence of freely diffusing FDH (curve 1). Upon the addition of increasing fructose concentrations (curve 2, 3, 4 and 5), a clear, substrate dependent catalytic current occurs, starting at about 100 mV vs. Ag/AgCl (1 M KCl). This indicates that the free enzyme reduces cyt c efficiently although it is trapped in a surface confined state. Plotting the catalytic currents over the fructose concentration reveals the behaviour of a typical enzymatic reaction following a Michelis-Menten-type kinetics (Figure 2B). Thus, an apparent (app) Km value of approx. 0.5 mM can be determined for the reaction of FDH with fructose as electron donating substrate and immobilized cyt c as an electron acceptor. It can be also shown that the biocatalytic reaction can be controlled by the addition of different FDH concentrations (under substrate-saturated conditions), as shown in Figure S1 in the supplement. The behaviour of commercially provided (Sigma-Aldrich) FDH and non-commercially produced FDH (isolated from Gluconobacter japonicus) was tested with cyt c monolayers at pH 7.0. A similar behaviour in cyt c reduction was found for both enzyme stocks, despite the marginally higher catalytic currents reached with the non-commercial enzyme. Since FDH has been reported to be inactivated by precipitation in the absence of stabilizing detergents,19 control experiments were performed in Triton X®-100 (TX)-containing and TX-free buffer. Here a decrease in the catalytic current was observed upon addition of the detergent (Figure S2). It has also been found that the enzyme remains active, even in TXfree buffer, at least in the period of the measurements. In order to create favourable conditions for the investigation of proteinprotein interaction and to minimize the risk of interference of

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Figure 2. Cyclic voltammograms of a cyt c monolayer electrode (AuE/MU:MUA/cyt c) with FDH (4 U/ml) in solution (5 mM KPi buffer, pH 7.0, SR = 2 mV/s). (A) Cyt c monolayer with FDH in substrate-free buffer (1) and upon addition of increasing fructose concentrations: 1.0 mM (2), 6.6 mM (3), 14.6 mM (4), 24.6 mM (5). (B) Plot of the percentage catalytic activity obtained at +200 mV vs. Ag/AgCl as a function of the fructose concentration. Here FDH isolated from Gluconobacter japonicus was used.

the detergent with the electrode, all electrochemical measurements were performed in buffer without TX addition. (It has to be added here that small amounts of TX are present since the detergent is part of the enzyme stock.) Acidic pH. Given that the pH optimum of the enzyme was reported to be between 4.0 and 4.5 and the UV/Vis study with both bio-molecules in solution also indicate that FDH reduces cyt c efficiently in this pH range, ET between FDH and immobilized cyt c was studied in acidic media. At pH 4.5 only a very small catalytic current was observed (supporting information, Figure S3). Going towards a slightly higher pH value of 5.0, the ET between FDH and cyt c seems to be improved (Figure 3A). A clear catalytic response was detected upon addition of substrate. However, the maximum current values are moderate compared to pH 7.0. The dependence of the ET reaction rate on the fructose concentration was analysed and is summarized in Figure 3B. An app Km value of about 2.6 mM was determined at pH 5.0. Here, the bio-electrocatalytic behaviour is clearly different from the one observed in media of neutral pH: (i) the found Km value is higher than the one determined for the same reaction at neutral pH and (ii) a lower maximum catalytic current was observed. This indicates that the ET pathway, which proceeds at neutral pH, enables a higher current flow at lower substrate concentrations. Therefore, it is suggested to be more efficient

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tion were performed under the same conditions. Here, no catalytic current was observed upon the addition of fructose. It can be concluded that DET between FDH and a MU:MUA modified electrode is not efficient under the present conditions, suggesting that ET proceeds via the redox protein cyt c. To investigate the influence of FDH adsorption on the redox behaviour of the cyt c monolayer, cyclic voltammograms of cyt c-FDH electrodes were recorded at a 100 mV/s before substrate addition and after scanning in the presence of the substrate. After FDH adsorption the cyt c-FDH electrodes showed a decreased cyt c redox activity (diminished peak currents) - compared to the situation before - as well as a shift of the typical Ef of adsorbed cyt c (i.e. 0 mV vs. Ag/AgCl, 1 M KCl) towards -25 to -30 mV (vs. Ag/AgCl, 1 M KCl) (supporting information, Figure S5). This suggests that the protein-protein binding between cyt c and FDH may affect cyt c`s redox properties. But it may also lead to the desorption of cyt c. Repeating a 100 mV/s scan after the study of biocatalysis showed a recovery of the initial cyt c signal, indicating the partial desorption of FDH on the one hand and the stability of the cyt c monolayer on the other hand. Desorption of the enzyme may be also an explanation for the moderate catalytic activity found. It can be concluded that the substrate-induced ET between FDH and cyt c is feasible even with both proteins confined on an electrode surface. Utilizing this finding and aiming on an increased catalytic response as well as a stable protein architecture, multiple layers of cyt c and FDH were built up on electrodes. Characteristics of cytochrome c:FDH multilayer systems. In order to enhance the catalytic response of the cyt c-FDH electrode, a multilayer assembly of FDH and cyt c was created by the help of a natural polymer DNA and applying the layerby-layer technique. Following the idea of a close contact between the catalytic bio-molecule and the redox protein, the coassembly of cyt c and FDH was addressed. The key point of this bio-macromolecular architecture is the incorporation of the enzyme in a matrix of the electron shuttling redox protein. This is based on the self-exchange capability of cyt c which can be retained even when immobilised by adsorption in multiple layers on electrodes.14,38,39 Cyt c acts consequently as reaction partner of FDH, but also as wiring agent connecting the enzyme molecules with the electrode by repeated interprotein electron transfer steps. Addressing the stabilization of this bi-protein construction, the negatively charged DNA was used as second building block. DNA and a mixture of cyt c and FDH were alternately deposited on a cyt c monolayer electrode creating the following multilayer system: Au/MU:MUA/cyt c/[DNA/cyt c:FDH]n(n = 3, 5 or 7). It has to be emphasized here that DNA acts only as a structural support to bind a large amount of cyt c.40 This is based on a rather strong interaction in acidic solutions which is mainly of electrostatic nature and has been elucidated previously.41 The molecule has no redox activity in the potential range tested here. Characterizing such a protein multilayer assembly, two issues had to be addressed: (i) Can cyt c and the 140-kDa FDH be co-immobilized on an electrode surface and (ii) is an ET reaction between FDH and cyt c possible within multilayered architectures with DNA?

than the one occurring in acidic media. However, it can also be observed that at high fructose concentrations the electron withdrawal from the reduced enzyme limits the ET process in both systems. (A)

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Figure 3. Cyclic voltammograms of a cyt c monolayer electrode (AuE/MU:MUA/cyt c) with FDH (4 U/ml) in a solution (20 mM KPi buffer, pH 5.0, SR = 2 mV/s). (A) Cyt c monolayer with FDH in the solution (1) and upon addition of increasing fructose concentrations: 0.5 mM (2), 5.0 mM (3), 15.0 mM (4), 45.0 mM (5). (B) Plot of the relative catalytic current obtained at 200 mV as a function of the fructose concentration. Here FDH provided by Sigma was used.

Investigation of FDH bound to a cytochrome c monolayer. In a next step, both proteins were immobilized by direct adsorption of FDH on a cyt c monolayer electrode. This strategy was inspired by a previous approach in which cyt c and sulfite oxidase were immobilized without the help of a polyelectrolyte or another building block, solely using the opposite net charges of the interaction partners.37 Given that FDH has an acidic isoelectric point of 5.0 and cyt c a basic one of 10, it was intended to create cyt c-FDH electrodes in the same manner. Therefore, cyt c monolayer electrodes were incubated in an FDH solution at pH 7.0. CVs was performed in protein-free buffer to investigate the cyt c-FDH electrodes in the absence and presence of fructose (Figure S4). Since the reaction between FDH and cyt c was found to be most efficient in media of neutral pH, the measurement was conducted at pH 7.0. Adding fructose resulted in a clear catalytic oxidation current, which started at -100 mV and reached a maximum level of 0.7 nA at +200 mV vs. Ag/AgCl (1 M KCl). Since it was also reported that adsorbed FDH is able to interact with modified gold electrodes directly,22 control measurements with MU:MUA electrodes incubated in FDH solu-

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Addressing the issue of co-immobilization, the mixed protein assembly was first analysed in a surface plasmon resonance (SPR) flow system. Therefore, a SPR Au-chip was modified with a MU/MUA self-assembled monolayer and a cyt c monolayer at pH 7.0. After a buffer exchange to 0.5 mM KPi buffer pH 5.0, DNA and the protein mixture were flushed over the chip surface. Figure 4 shows the stepwise assembly of both components with a cyt c:FDH ratio of 24:1. A clear and defined adsorption from the mixed protein solution was found with only a small amount of loosely bound material, which can be removed in the subsequent washing step. The sensorgram also indicates that the adsorption time of 10 min is not sufficient to saturate the system with cyt c:FDH completely. Since a well-defined and reproducible deposition of proteins is already reached within this period, the incubation was not prolonged and analogously used for the subsequent electrode preparation. It has to be mentioned here that the joint immobilisation is possible since FDH possesses not the same charge as cyt c at this pH (pIs: cyt c - 10, FDH - 5) – a feature which hinders for example the integration of PQQ-GDH into such multilayers systems.45 Interestingly the deposition of DNA on the mixed protein layer resulted only in a small change of the resonance units. Nevertheless, it can be concluded that the negatively charged DNA did adsorb, since the assembly of a subsequent protein layer was possible. It has to be added here that DNA deposition on cyt c could be followed in previous studies using quartz crystal microbalance (QCM) as a detection method.16,40 In these studies a clear mass accumulation was found upon DNA adsorption. This can be explained by the fact that QCM also detects surface-bound water (which DNA is rich of) and SPR does not. 1x

2x

3x

4x

(A) 20.0

Current I (nA)

10.0

8000

0.1

0.2

Current I (nA)

27nA

0.0

2 1 -0.2

-0.1

0.0

0.1

0.2

Potential E (V)

Figure 5. Cyclic voltammograms of multilayer electrodes comprising 5 bilayers of DNA and cyt c:FDH (Au/MU:MUA/ cyt c/[DNA/(cyt c:FDH)]5) which were constructed using different cyt c:FDH ratios of (A) 24:1 and (B) 5:1 (5 mM KPi buffer, pH 7.0, SR = 2 mV/s). Modified electrode in the absence (1) and upon addition of 10 mM fructose (2). Here FDH isolated from Gluconobacter japonicus was used.

multilayer electrode are shown in Figure 5. Here, the surface density of redox-active cyt c was found to increase with the number of layers assembled reaching around 130 pmol/cm2 for five bilayers (Table 1). This high electro-activity verifies that the multilayer system can be constructed and is a clear hint for the efficient ET through the layered assembly based on cyt c-cyt c inter-protein ET. In comparison to the surface density of cyt c-DNA multilayers without enzyme incorporation (225 pmol/cm2 for five layers) the reached value was lower.

0 6000

0.0

20.0

-20.0

5000

4000

-0.1

(B) 40.0

DNA KPi pH 5.0 cyt c:FDH

2000

2 1

Potential E (V)

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-10.0

-0.2

5x

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4 nA 0.0

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20000

Response (RU)

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10000

Time t (s)

Figure 4. SPR sensorgram of the cyt c:FDH multilayer assembly, constructed on a MU:MUA modified gold chip with a cyt c monolayer, five bilayers of DNA and the cyt c:FDH mixture (ratio 24:1). The experiment was performed in a flow system under a constant flow rate of 1 µl/min, using 0.5 mM KPi buffer pH 5.0 for the assembly of the alternating DNA/cyt c:FDH layers. Here FDH isolated from Gluconobacter japonicus was used.

Table 1. Catalytic currents (determined at +200 mV vs Ag/AgCl) and cyt c surface concentration of cyt c:FDH multilayer electrodes (cyt c/FDH ratio 24:1) comprising 3, 5 and 7 bilayers. Number of layers

The multilayer assembly was found to be stable after the deposition of the final cyt c:FDH layer, since the SPR signal remained stable under buffer flow. Changing the buffer to 5 mM KPi buffer pH 7.0, which was used for the conduction of CV, also no major change of the signal was recorded, indicating the stability of the system even at neutral pH. Preparing the DNA-protein architecture on a thiol-modified electrode with a monolayer of cyt c, CV measurements were performed. Cyclic voltammograms of a [DNA/(cyt c:FDH)]5

3 5 7

Catalytic current (nA) 0.5 4.4 8.3

Cyt c surface conc. (pmol/cm2) 15 127 222

This can be considered as first indication for the successful deposition of FDH together with cyt c. Upon the addition of fructose to the buffer solution a catalytic response was ob-

6


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served for this kind of bi-protein protein electrodes. This confirms the immobilization of active FDH and cyt c from the mixed propr tein solution during the preparation. Moreover, the magnitude of this response was dependent on the number of layers assembled (Table 1). Scheme 1 depicts the ET mechanism, which may be concluded from thee catalytic response observed in the CV measmea urements. It suggests that the reduced FDH may donate elecele trons to cyt c in its close environment (after the catalytic elecele tron extraction from the substrate). Then, electrons are shuttled towards the electrode via cyt c-cyt c self-exchange. exchange. In the final step a cyt c molecule ecule immobilised on the MU:MUA SAM exchange electrons with the electrode, generating an oxidative catalytic current.

modification, the FDH-cyt c interaction is limiting the system by electron withdrawal from the reduced red enzyme towards the electrode. In order to study the influence of the cyt c:FDH concentration ratio in the assembly solutions on the behaviour behavio of the biprotein electrode, [DNA/(cyt c:FDH)]5 multilayer electrodes were prepared using cyt c:FDH :FDH ratios of 100:1, 24:1 and 5:1. A lower relative amount (100:1) of FDH turned out to be not beneficial for the defined catalysis, however by increasing the FDH content (ratio 5:1) an even more efficient bio bioelectrocatalytic trocatalytic behavior was found compared to the preparation from a 24:1 solution. This result is illustrated in Figure 5B.. Here, still the concept of multilayer formation works, as can be concluded from the cyt c surface concentration found (about 100 pmol/cm2). This finding may again illustrate that the multilayer system is not limited by the cyt c – cyt c electron exchange allowing the defined detection of the anaan lyte dependent conversion at the enzyme. A further raise in the FDH concentration limits the multilayer formation and only small cyt c concentrations ations have been observed. The robustness of the cyt c:FDH c multilayer electrodes during measurement and storage was also studied. The archite architectures were found to be resistant against mechanic mechanical stress caused by stirring and buffer change. After Aft three days of storage in 0.5 mM KPi buffer pH 5.0 at 4 °C, an average activity

Scheme 1. Schematic illustration of electron transfer steps occurring in a cyt c-FDH FDH multilayer electrode in the presence of fructose.

(A)

3.5 mM

2.5 mM 2.8 2.6

Current I (nA)

At this point one may compare the pure protein-based protein approach demonstrated here with a redox polymer with defined redox centres (e.g. .g. Os complexes). Also for such polymers electrons are transported by a hopping mechanism between the redox centres. Diffusion coefficients for this process were found to depend on the film structure, but are in the range 10-9 – 10-11 cm2s-1 resulting in exchange rate constants kex of 104 – 106 M-1s-1.46-48 Although lower in the efficiency of self exe change (kex ~ 104 M-1s-1)49 the cyt c approach allows still suff sufficient electron transport to result in fructose dependent catalytic currents. It has to be added that only at higher scan rates du during voltammetry the self-exchange exchange becomes limiting resulting in decreasing catalytic currents. Furthermore the cyt c based approach only relies on the use of biomolecules for the formaform tion of the sensing electrode. In order too investigate the sensitivity of the system towards fructose, amperometric measurements were performed. The multilayer system was found to be sensitive towards the subsu strate in a range of 0.005 mM to 10 mM and did not respond to fructose concentrations below 0.005 mM. Figure 6A depicts a section off the sensor response using a [DNA/(cyt c:FDH)]5 multilayer electrode during an ama perometric measurement, induced by addition of fructose in the range from 1.0 mM to 3.5 mM. For each concentration, concentration well-defined defined response behaviour was found. Plotting the rer corded catalytic currents over the fructose concentration rer sulted in a curve following the Michaelis-Menten Menten-type kinetics (Figure 6B). ). Saturation was already reached at lower mM concentrations i.e. app Km= 0.3 mM. This value is very close to the app Km value obtained with freely diffusing FDH (0.5 mM), indicating ng again that in the DNA-supported DNA surface

1.5 mM

2.4 2.2

1.0 mM

2.0 1.8 1.6 150

200

250

300

350

400

Time t (s)

(B) 4.0

Catalytic current (nA)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


3.0

2.0

1.0

0.0 0

2

4

6

8

10

12

14


Figure 6. Electrochemical ochemical characterization of [DNA/(cyt c:FDH)]5 bilayer electrodes (Au/MU:MUA/cyt c/[DNA/(cyt [DNA/(cyt c:FDH)]5) at pH 7.0 (5 mMKPi buffer). ). (A) Amperometric measurement of the modified elece trode with stepwise increasing fructose concentrations from 0.01 mM to 21 mM. (B) Plot of the catalytic currents obtained from three independentamperometric amperometric measurements (E =+0.2 = V vs. Ag/AgCl, 1 M KCl) of [DNA/(cyt c:FDH)]5multilayer electrodes at different fructose concentrations.

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This material is available free of charge via the Internet at http://pubs.acs.org.

of about 75 % compared to the first day was retained, while measurements after 11 days still showed a response of 55 %. This indicates that the co-entrapment of FDH with in a multilayer system preserves the enzyme activity despite the absence of a stabilizing detergent, such as TX.

AUTHOR INFORMATION Corresponding Author

CONCLUSIONS The ET reaction of the flavin-dependent enzyme FDH with the redox protein cyt c was investigated in solution and in a surface confined state. In solution, two different pH optima were found for the reaction at pH 4.0 and 7.0. When cyt c was adsorbed on an electrode surface, ET with freely diffusing FDH was found to proceed at pH 5.0 and at pH 7.0. However, the ET reaction seems to be more efficient at neutral pH, since higher catalytic currents were obtained. The behaviour at pH 7.0 and 5.0 may suggest that two different ET pathways occur. Our experiments also indicate that inter-protein ET is feasible when one partner is in the immobilized state. Furthermore, it was demonstrated that the cyt c:FDH reaction can be exploited with both proteins fixed to the electrode surface. Since the direct adsorption of FDH on a cyt cmonolayer resulted in moderate catalytic activity only, the construction of a cyt c:FDH multilayer architecture was addressed with the help of the bio-polymer DNA. A well-defined layered assembly of DNA/(cyt c:FDH) architectures was shown by SPR and transferred to a thiol-modified gold electrode surface. Performing CV measurement the multilayer electrode exhibited an increased cyt c concentration in comparison to a cyt c monolayer system and a clear catalytic current in the presence of fructose. This current was found to increase with the number of cyt c:FDH-layers deposited and a decreasing ratio cyt c:FDH used in the assembly process during the electrode preparation. Even when both proteins were fixed to a surface in a multilayer architecture, an efficient ET was enabled between the reaction partners, resulting in defined responses towards fructose concentrations. The study shows that proteins can be arranged artificially in 3D-architectures to result in new functional systems following natural examples. Multiple layer deposition and multi-protein electron transfer steps are the basic construction and operation principles here. Such architectures are interesting for sensing applications since not only one enzyme can be incorporated (as also shown e.g. for BOD8 or SOx13,37), the sensing properties can be tuned by number of layers as well as the relative ratio of cyt c and the enzyme and because of a good storage stability of the prepared all-biomolecule-electrodes. In addition, application can be seen in other fields such as photobioelectrocatalysis since even more complex biomolecules such as the supercomplex photosystem I can be incorporated into a layered cyt c matrix resulting in efficient light to current conversion50.

*E-mail for Fred Lisdat: [email protected].

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT Financial support by the DFG project Li 706/7-1is gratefully acknowledged.

REFERENCES (1) Berg, J. M.; Tymoczko, J. L.; Stryer, L. Biochemistry, 7th ed.; W.H. Freeman: New York, 2012; p 41, 43, 48, 1054. (2) Taniguchi, I.; Toyosawa, K.; Yamaguchi, H.; Yasukouchi, K. J. Electroanal. Chem. 1982, 140, 187-193. (3) Song, S.; Clark, R. A.; Bowden, E. F.; Tarlov, M. J. J. Phys. Chem. 1993, 97, 6564-6572. (4) Ge, B.; Lisdat, F. Anal. Chim. Acta 2002, 454, 53-64. (5) Armstrong, F. A.; Hill, H. A. O.; Walton, N. J. Accounts Chem. Res. 1988, 21, 407-413. (6) Feifel, S. C.; Ludwig, R.; Gorton, L.; Lisdat, F. Langmuir 2012, 28, 9189-9194. (7) Sarauli, D.; Tanne, J.; Xu, C. G.; Schulz, B.; Trnkova, L.; Lisdat, F. Phys. Chem. Chem. Phys, 2010, 12, 14271-14277. (8) Lisdat, F.; Dronov, R.; Moehwald, H.; Scheller, F. W.; Kurth, D. G. Chem. Commun. 2009, 3, 274-283. (9) Jin, W.; Wollenberger, U.; Bier, F. F.; Makower, A.; Scheller, F. W. Bioelectroch. Bioener. 1996, 39, 221-225. (10) Jin, W.; Wollenberger, U.; Kargel, E.; Schunck, W. H.; Scheller, F. W. J. Electroanal. Chem. 1997, 433, 135-139. (11) Sarauli, D.; Ludwig, R.; Haltrich, D.; Gorton, L.; Lisdat, F. Bioelectrochemistry 2012, 87, 9-14. (12) Katz, E.; Willner, I. J. Am. Chem. Soc. 2003, 125, 68036813. (13) Spricigo, R.; Dronov, R.; Rajagopalan, K. V.; Lisdat, F.; Leimkühler, S.; Scheller, F. W.; Wollenberger, U. Soft Matter 2008, 4, 972-978. (14) Spricigo, R.; Dronov, R.; Lisdat, F.; Leimkühler, S.; Scheller, F. W.; Wollenberger, U. Anal. Bioanal. Chem. 2009, 393, 225-233. (15) Feifel, S. C.; Kapp, A.; Ludwig, R.; Gorton, L.; Lisdat, F. RSC Advances 2013, 3, 3428-3437. (16) Wettstein, C.; Möhwald, H.; Lisdat, F. Bioelectrochemistry 2012, 88, 97-102. (17) Cooney, M. J.; Svoboda, V.; Lau, C.; Martin, G.; Minteer, S. D. Energ. Environ. Sci. 2008, 1, 320-337. (18) Wu, Y. H.; Hu, S. S. Microchim. Acta 2007, 159, 1-17. (19) Ameyama, M.; Shinagawa, E.; Matsushita, K.; Adachi, O. J. Bacteriol. 1981, 145, 814-823. (20) Kawai, S.; Goda-Tsutsumi, M.; Yakushi, T.; Kano, K.; Matsushita, K. Appl. Environ. Microb. 2013, 79, 1654-1660. (21) Kawai, S.; Yakushi, T.; Matsushita, K.; Kitazumi, Y.; Shirai, O.; Kano, K. Electrochem. Commun. 2014, 38, 28-31. (22) Khan, G. F.; Shinohara, H.; Ikariyama, Y.; Aizawa, M. J. Electroanal. Chem. 1991, 315, 263-273. (23) Kamitaka, Y.; Tsujimura, S.; Setoyama, N.; Kajino, T.; Kano, K. Phys. Chem. Chem. Phys. 2007, 9, 1793-1801.

ASSOCIATED CONTENT Supporting Information Available Cyclic voltammograms of the effect of the enzyme activity and the presence of TX on the catalytic current, the bio-electrocatalytic reaction between a cyt c monolayer and FDH at pH 4.0 as well as FDH adsorbed directly on a cyt c monolayer.

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(24) Ikeda, T.; Matsushita, F.; Senda, M. Biosen. Bioelectron. 1991, 6, 299-304. (25) Marcinkeviciene, J.; Johansson, G. FEBS Letters 1993, 318, 23-26. (26) Sasaki, Y.; Sugihara, T.; Osakai, T. Anal. Biochem. 2011, 417, 129-135. (27) Khan, G. F.; Kobatake, E.; Shinohara, H.; Ikariyama, Y.; Aizawa, M. Anal. Chem. 1992, 64, 1254-1258. (28) Kinnear, K. T.; Monbouquette, H. G. Anal. Chem. 1997, 69, 1771-1775. (29) Bassi, A. S.; Lee, E.; Zhu, J. X. Food Res. Int. 1998, 31, 119-127. (30) Tkac, J.; Svitel, J.; Vostiar, I.; Navratil, M.; Gemeiner, P. Bioelectrochemistry 2009, 76, 53-62. (31) Tamaki, T. Top. Catal. 2012, 55, 1162-1180. (32) Tominaga, M.; Shirakihara, C.; Taniguchi, I. J. Electroanal. Chem. 2007, 610, 1-8. (33) Tominaga, M.; Nomura, S.; Taniguchi, I. Biosens. Bioelectron. 2009, 24, 1184-1188. (34) Murata, K.; Suzuki, M.; Kajiya, K.; Nakamura, N.; Ohno, H. Electrochem. Commun. 2009, 11, 668-671. (35) Sarauli, D.; Wettstein, C.; Peters, K.; Schulz, B.; Fattakhova-Rohlfing, D.; Lisdat, F. ACS Catalysis 2015, 5, 20812087. (36) Ferapontova, E. E.; Gorton, L. Bioelectrochemistry 2005, 66, 55-63. (37) Dronov, R.; Kurth, D. G.; Moehwald, H.; Spricigo, R.; Leimkuehler, S.; Wollenberger, U.; Rajagopalan, K. V.; Scheller, F. W.; Lisdat, F. J. Am. Chem. Soc. 2008, 130, 1122-1123.

(38) Feifel, S. C.; Kapp, A.; Lisdat, F. Langmuir 2014, 30, 5363-5367. (39) Wegerich, F.; Turano, P.; Allegrozzi, M.; Mohwald, H.; Lisdat, F. Langmuir 2011, 27, 4202-4211. (40) Sarauli, D.; Tanne, J.; Schaefer, D.; Schubart, I. W.; Lisdat, F. Electrochem. Commun. 2009, 11, 2288-2291. (41) Wettstein, C.; Kyne, C.; Doolan, A. M.; Mohwald, H.; Crowley, P. B.; Lisdat, F. Nanoscale 2014, 6, 13779-13786. (42) van Gelder, B.; Slater, E. C. Biochim. Biophys. Acta 1962, 58, 593-595. (43) Bard, A. J.; Stratmann, M. Encyclopedia of electrochemistry; Wiley-VCH: Weinheim, 2007; p 402. (44) Kepplinger, C.; Lisdat, F.; Wollenberger, U. Langmuir 2011, 27, 8309-8315. (45) Wettstein, C.; Möhwald, H.; Lisdat, F. Bioelectrochemistry 2012, 88, 97-102. (46) Pickup, P. G.; Murray, R. W. J. Am. Chem. Soc. 1983, 105, 4510-4514. (47) Sosnoff, C. S.; Sullivan, M.; Murray, R. W. J. Phys. Chem. 1994, 51, 13643-13650. (48) O´Mullane, A. P.; Macpherson, J. V.; Unwin, P. R.; Cervera-Montesinos, J.; Manzanares, J. A.; Frehill, F.; Vos, J. G. J. Phys. Chem. B 2004, 108, 7219-7227. (49) Feifel, S. C.; Lisdat, F. J. Nanobiotechnol. 2011, 9:59 (50) Stieger, K. R.; Feifel, S. C.; Lokstein, H.; Lisdat, F. Phys. Chem. Chem. Phys. 2014, 16, 15667-15674.

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C urrent I

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Interaction of Flavin-Dependent Fructose Dehydrogenase with

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