Determination of the pH Dependent Redox Potential of Glucose

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Determination of the pH dependent redox potential of glucose oxidase by spectroelectrochemistry Stephan Vogt, Marcel Schneider, Heiko Schäfer-Eberwein, and Gilbert Nöll Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac501289x • Publication Date (Web): 09 Jul 2014 Downloaded from http://pubs.acs.org on July 17, 2014

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Analytical Chemistry

Determination of the pH dependent redox potential of glucose oxidase by spectroelectrochemistry Stephan Vogt,1 Marcel Schneider,2 Heiko Schäfer-Eberwein,2 Gilbert Nöll1* 1

Nöll Junior Research Group, University of Siegen, Adolf-Reichwein-Straße 2, D-57068 Siegen,

Germany 2

Institute for High Frequency and Quantum Electronics, University of Siegen, Hölderlinstraße 3,

D-57076 Siegen, Germany. KEYWORDS: biosensor, biofuel cell, glucose oxidase (GOx), spectroelectrochemistry, flavins and flavoproteins

ABSTRACT The pH dependent redox potential of the oxidoreductase glucose oxidase (GOx) from Aspergillus niger, which is the most frequently applied enzyme in electrochemical glucose biosensors and biofuel cells, was measured between pH 4.5 and 8.5 using UV/Vis spectroelectrochemistry. In the entire pH range under investigation, the FAD cofactor of GOx changed directly from the oxidized quinone to the doubly reduced hydroquinone. No stable semiquinoid species could be detected if electrochemical equilibrium was reached. From the pH dependency of the GOx redox potential a pKa of 7.2 has been determined for the GOx flavohydroquinone. At pH values ≤ pH 6.0 a dependency of the reduction mechanism and the

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GOx redox potential on the presence of halides, especially on Cl-, was observed. For the development of glucose biosensors and glucose biofuel cell anodes working at physiological or neutral pH, the GOx redox potentials at pH 7.4 and pH 7.0 are of main interest. Here values of E1/2 pH 7.4 = -97 ± 3 mV and E1/2 pH 7.0 = -80 ± 4 mV have been determined. Introduction Glucose oxidase (GOx) is a homodimeric flavoprotein, which catalyzes the oxidation of

β-D-glucose to δ-gluconolactone.1 During this process, the tightly bound cofactor flavin adenine dinucleotide (FAD) takes up two electrons (as well as one or two protons), which are used in nature to reduce dioxygen to hydrogen peroxide.1 With its high specificity for monosaccharides,1,2 the easy accessibility from different fungi and insects,1 and turnover rates up to 5000 s-1,3 GOx meets many important requirements for applications in glucose biosensors.2 Furthermore, the enzyme has been thoroughly applied as catalyst at glucose biofuel cell anodes,4,5 in enzyme-based logic gates,6 and as model enzyme for several strategies to contact enzymes electrochemically.7-11 Up to now, more than 6000 papers have been published on GOx in the context of glucose biosensors or biofuel cells (SciFinder Scholar 2014). In order to achieve efficient electron transfer (ET) between the cofactor FAD (which is deeply buried in the apoenzyme) and the electrode, different types of electron shuttles, i.e. redox mediators have been used. For optimum performance, the redox potential of the mediator has to be adjusted with respect to that of the enzyme.12 Thus, the exact knowledge of the pH dependent GOx redox potential, in the pH range relevant for the development of electrochemical glucose biosensors and biofuel cells, is of high interest. Nevertheless only a few papers with focus on this topic have been published up to now. Since the published values differ by several hundred millivolts, the redox potential of GOx is still under debate. By anaerobic redox titration with sodium dithionite


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Walter determined the redox potential to be -144 ± 5 mV vs. NHE at pH 7.0.13 During his work on the development of glucose biofuel cells, in 2004 Heller supposed the GOx redox potential to be -360 mV vs. Ag/AgCl at pH 7.2 (the chloride concentration is not further specified).12 Assuming that an Ag/AgCl/KCl (1 M) reference electrode has been used by Heller, this corresponds to a redox potential of -125 mV vs. NHE. Some years later Heller and Feldman suggested a value of -48 mV vs. NHE for pH 7.2 based on literature values.3 In several recent publications, the direct electrochemical investigation of glucose oxidase is described. However, the measured values differ very much depending on the type of the working electrode. Zhang et al. published a redox potential of -207 mV vs. NHE at pH 7.0 using nitrogen-doped carbon hollow spheres,14 whereas Alwarappan et al. measured +389 mV vs. NHE at pH 7.0 on a graphene nanosheet modified gold electrode.15 For deglycosylated GOx at pH 7.4 a value of -280 mV vs. NHE was estimated by Mano and coworkers.16 All redox potential values discussed so far correspond to the overall reduction (and subsequent reoxidation) of the GOx bound flavin cofactor FAD from the fully oxidized quinone to the doubly reduced hydroquinone state. The flavin reduction follows an ece(c) mechanism, i.e. a first electrochemical reaction (electron transfer) leading to the semiquinone radical anion is followed by a chemical reaction (protonation). The resulting neutral semiquinone radical can be reduced a second time (second e-step) leading to a hydroquinone anion, which, depending on pH, will also be protonated.17-19 For free flavins in aqueous solution the redox potential of the second electron transfer (ET) reaction is less negative than that of the first. Therefore, both redox processes are overlapping, and only the oxidized and the doubly reduced flavin states are stable. In some flavoenzymes also the singly reduced flavin states, i.e. the neutral semiquinone radical or the semiquinone radical anion, can be stabilized by interaction between flavin and


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apoenzyme.20 Also for GOx the neutral flavosemiquinone radical with a broad absorption band, showing a maximum at λmax = 560 nm, or the flavosemiquinone radical anion with an absorption maximum of λmax = 490 nm have been observed when GOx was reduced photochemically by irradiation with blue-light in the presence of EDTA at pH 5.3 or pH 10.3, respectively.17,20,21 However, the photochemical reduction of flavoproteins by irradiation with blue-light in the presence of EDTA does not require electrochemical equilibrium.17,18 At pH 5.3, Stankovich et al. found a redox potential of -63 ± 11 mV and -65 ± 7 mV vs. NHE for the first and the second reduction step, respectively, using photochemical reduction with EDTA.21 By anaerobic redox titrations with sodium dithionite they determined the first and second redox potential of GOx to be -200 ± 10 mV and -240 ± 5 mV vs. NHE at pH 9.3.21 Thus, the determination of the GOx redox potential, using an experimental technique, which allows the determination of the overall redox potential and provides additional information about the possible formation of thermodynamically stable intermediates, is of strong interest. In this work we present the determination of the pH dependent GOx redox potential and elucidate mechanistic details of the enzyme redox chemistry by UV/Vis spectroelectrochemistry. Using this technique also the singly reduced flavin radicals can be detected, if they are stable within the time scale of the experiment. We decided to carry out our measurements in the range between between pH 4.5 and pH 8.5. Acidic pH values are of interest in biofuel cell research e.g. when a GOx modified anode is combined with a laccase modified cathode for dioxygen reduction (depending on its origin the enzyme laccase has a pH optimum around pH 5).12,22 When bilirubin oxidase (with a pH optimum between pH 3 and pH 8, depending on substrate and organism) is used at the cathode, glucose biofuel cells can be applied at neutral pH or even at pH 7.4 (physiological pH).23 Since pH 7.4 is the pH of human blood, this


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pH value is relevant for the development of glucose biosensors as well as potentially implantable glucose biofuel cells.3 Significantly more basic pH values are of less importance, since the highest catalytic activity of GOx is expected between pH 4.0 and pH 7.0 with an optimum around pH 5.5).24,25 While Massey and Palmer reported that GOx is stable (for at least 20 hours) and also catalytically active between pH 3.5 and pH 10.3,20 other groups found GOx to be instable below pH 4.5 and above pH 7.5.1,13 For our measurements we used a low volume spectroelectrochemical cell, which has been described previously by Bistolas et al. with minor changes.26 In order to establish electrochemical equilibrium, the spectroelectrochemical measurements were done in the presence of different redox mediators depending on pH.27,28 Experimental Section GOx from Aspergillus niger was purchased from Serva Electrophoresis (Heidelberg, Germany), flavin adenine dinucleotide (FAD) from TCI Europe N.V. (Zwijndrecht, Belgium), and flavin mononucleotide (FMN) from Sigma-Aldrich (Steinheim, Germany). Measurements were done with an AMEL model 2049 potentiostat (Amel Electrochemistry, Milan, Italy), an Avantes Avalight DH-S-BAL light source, and an AvaSpec ULS 3648 photospectrometer connected via Avantes optical fibers (Avantes BV, Apeldoorn, The Netherlands). A homemade spectroelectrochemical cell with an inner volume of less than 100 µL, similar to that reported by Bistolas et al.,26 was used (see Figure S5 in the supporting information for details). The inner diameter of the gold capillary working electrode was 0.25 mm. A LabView routine was programmed to control the setup with a National Instruments NI USB-6211 card (National Instruments, Austin, TX, USA). To protect GOx from continuous light irradiation, the shutter was only opened for several milliseconds when spectra were recorded. For the UV/VIS


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measurements of FAD and oxidized GOx, a PerkinElmer Lambda 750 photospectrometer (PerkinElmer, Waltham, MA, USA) was used. Spectroelectrochemical measurements of GOx were done between pH 4.5 and pH 8.5 with enzyme concentrations between 75 µM and 150 µM (based on the molar mass of homodimeric glucose oxidase; M= 160 kDa). Measurements were performed in 0.1 M buffer (depending on pH sodium citrate, potassium phosphate, or tris(hydroxymethyl)-aminomethan (TRIS)-buffer were used) with 0.1 M potassium chloride and 5 mM magnesium chloride. The buffers were thoroughly deoxygenated by purging with argon and subsequent degassing under vacuum to prevent evolution of gas in the gold capillary working electrode. The composition of the sample solutions at each pH including the used mediator mixtures can be found in Table S2 of the supporting information. To ensure that the results of our experiments were not influenced by artifacts caused by the mediator mixture, the concentrations of the employed mediators were varied throughout the series of redox titrations. All potentials were measured against an Ag/AgCl/KCl reference electrode with an overall chloride concentration of 0.11 M (the electrolyte contained 0.1 M KCl and 5 mM MgCl2). For an Ag/AgCl/KCl (0.1 M) reference electrode a potential of 288 mV vs. NHE at 25 °C is published.29,30 Since the overall chloride concentration was 0.11 M, a potential of 286 mV vs. NHE was calculated based on the concentration (activity) dependent term of the Nernst equation.31 All values are given vs. NHE. Titration curves for GOx were obtained by plotting the absorption at 456 nm of the individual spectra against the corresponding electrode potential. Measurements of GOx and reference compounds were evaluated by linearization of the redox titration curves in a semilogarithmic plot as described by Dutton.27 In order to check the accuracy of the redox potentials determined by this method we also evaluated our measurements with


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singular value decomposition (SVD). While the basic procedure of SVD has been described by Hendler and Shrager,32,33 we used the commercial software SpecFit 32 (Spectrum Software, Marlborough, USA) to determine the redox potential by SVD. The redox potentials determined by SVD (see Table S1 in the supporting information) are in agreement (within the standard deviation) with the redox potentials determined by linearization (see Table 1). To ensure the proper function of the spectroelectrochemical setup, FAD was measured before and after each spectroelectrochemical measurement of GOx as reference compound. Redox titrations of FAD, which can be reduced and oxidized directly at the electrode surface (in the absence of redox mediators), took approximately 45 minutes per measurement, whereas the spectroelectrochemical measurements of GOx took considerably more time. Results and Discussion The measured redox potentials for our reference compound FAD at pH 7.0 varied between -197 mV and -220 mV. From all measured values a mean value of -207 mV including a standard deviation of 7 mV was calculated, which is up to 12 mV less negative than published values (a redox potential of -219 mV has been determined by anaerobic chemical redox titration,34 -213 mV by polarography,35). The mean values including their standard deviations measured for FAD at different pH are summarized in Table 1. The redox potential of free FAD showed a pH dependence as expected for free flavins.36 At pH 7.0 we measured also the redox potential of the flavin cofactor flavin mononucleotide (FMN). For FMN at pH 7.0 a redox potential of -208 ± 2 mV was measured, which is in agreement with the published values of -205 mV, -210 mV, and -211 mV.34,35,37

From these control experiments we concluded that our

spectroelectrochemical setup was working in a proper way.


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Due to the particular importance of the GOx redox potential at pH 7.4 for glucose biosensors, we started our measurements on GOx at this pH value. During each experimental run a complete redox cycle ranging from the oxidized to fully reduced enzyme and back was measured. The experimental conditions were optimized until the initial spectra of the oxidized enzyme could be recovered after reoxidation at the end of the complete redox cycle (indicating that the enzyme was still intact). Furthermore, the redox mediator mixtures and equilibration times (i.e. the delay time after each potential step before a spectrum is recorded) were optimized thoroughly to achieve electrochemical equilibrium, which is indicated by merging titration curves for reduction and reoxidation. In order to achieve electrochemical equilibrium, rather long equilibration times were necessary. A single redox cycle (including reduction and reoxidation) took up to 60 hours. The use of a mediator mixture composed of ammonium iron(III) oxalate and cyanomethyl viologen turned out to be very efficient. In Figure 1A only the spectra collected during the stepwise reduction of GOx under optimized conditions are shown. For the complete spectroelectrochemical measurement the titration curves, i.e. the potential dependent absorption values at 456 nm (absorption maximum of oxidized GOx) collected during reduction and reoxidation, are shown in Figure 1B. During reduction and reoxidation the spectra of GOx changed from those corresponding to the oxidized quinone state directly to those of the doubly reduced hydroquinone and back. There was no indication for the formation of a stable neutral semiquinone radical or radical anion. At pH 7.4, an overall redox

potential of

E1/2 pH 7.4 = -97 ± 3 mV was determined.


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Figure 1. A) UV/Vis-spectra collected during the stepwise reduction of GOx at pH 7.4. B) The corresponding titration curves at λ = 456 nm for reduction and subsequent reoxidation. Ammonium iron(III)-oxalate trihydrate (50 µM) and cyanomethyl viologen (50 µM) were used as redox mediators. Measurements were performed in 0.1 M phosphate buffer, containing 0.1 M KCl and 5 mM MgCl2.

Next, spectroelectrochemical measurements were carried out at pH 7.0 leading to a redox potential of E1/2 pH 7.0 = -80 ± 4 mV vs. NHE. Thereafter we continued our investigations at acidic pH in pH intervals of 0.5 down to pH 4.5. Finally we measured the GOx redox potential also at slightly basic pH values (between pH 7.4 and 8.5). Our measurements were limited to pH 8.5,


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since at more basic pH values the applied viologen redox mediators underwent significant degradation. It is well known that viologens decompose at basic conditions.38 The pH dependent GOx redox potentials including their standard deviations are summarized in Table 1. When the spectroelectrochemical measurements were carried out under optimized conditions reaching electrochemical equilibrium, no semiquinoid species could be observed in the entire pH range under investigation. In this context it is of particular interest to discuss the influence of the employed redox mediators. When we used only ammonium iron(III) oxalate as redox mediator at pH 5.0, we observed the formation of the neutral semiquinone radical form of GOx to a considerable amount as indicated by a broad absorption band between 530 nm and 650 nm (see Figure 2A). However, in the corresponding titration curves (see Figure 2B) there was still a small hysteresis indicating that electrochemical equilibrium was not yet reached. Therefore we tried to improve the experimental conditions using additional redox mediators. By adding the positively

charged

cyanomethyl

viologen

to

the

redox

mediator

mixture,

the

spectroelectrochemical measurements could be carried out in electrochemical equilibrium as indicated by merging titration curves shown in Figure 3B. As it can be seen from the spectral changes during the reductive half cycle (see Figure 3A) there was no indication for the formation of a semiquinone radical any more. Apparently the careful choice of a proper mediator mixture is of strong importance in order to establish electrochemical equilibrium. At nonequilibrium conditions the singly reduced semiquinone radical of GOx may accumulate. This observation might explain why the semiquinone radical (or radical anion) has been observed in previous work on GOx.20,21


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Figure 2. A) UV/Vis-spectra collected during the stepwise reduction of GOx at pH 5.0. B) The corresponding titration curves at λ = 456 nm for reduction and subsequent reoxidation. The broad absorption band between 530 nm and 650 nm in (A) is typical for the neutral flavosemiquinone radical. As it can be seen from the small hysteresis between the titration curves in (B), the system was not in electrochemical equilibrium. As redox mediator only ammonium iron(III) oxalate (100 µM) was used. Measurements were performed in 0.1 M citrate buffer containing 0.1 M KCl and 5 mM MgCl2.


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Figure 3. A) UV/Vis-spectra collected during the stepwise reduction of GOx at pH 5.0. B) The corresponding titration curves at λ = 456 nm for reduction and subsequent reoxidation. The absence of any hysteresis between the titration curves in (B) implies that the system was in electrochemical equilibrium. Here a mixture of the redox mediators cyanomethyl viologen (40 µM), phenazine methosulfate (50 µM) and ammonium iron(III) oxalate (500 µM) was used. Measurements were performed in 0.1 M citrate buffer with 0.1 M KCl and 5 mM MgCl2.

Next, the pH-dependency of the redox potentials of (free) FAD and GOx will be discussed in detail. According to Nernst a slope of -59 mV ∙ pH-1 value is expected at acidic pH for the overall reaction of the flavin redox center (Fl): Flox + 2e- + 2H+ ⇌ FlredH2,36 whereas a slope of only -30 mV · pH-1 is expected (for the reaction Flox + 2e- + H+ ⇌ FlredH-), if the


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flavohydroquinone stays deprotonated. Hence, it might be even possible to determine the pKa of the flavohydroquinone from a pH dependent determination of the redox potential. For the pKa of free FAD hydroquinone values of 6.2,39 6.3,40 6.5,41 6.7,34 and 6.835 have been reported. Plots of the FAD and GOx redox potentials vs. pH are depicted in Figure 4A and B, respectively.

Figure 4. Plot of the FAD (A) and GOx (B) redox potential vs. pH. A) For free FAD two distinct regions with different pH dependency of the redox potential were observed. Slopes of -50 mV · pH-1 and -27 mV · pH-1 were calculated by least squares fit from the values between pH 4.0 and pH 6.5 and between pH 7.4 and pH 8.8, respectively. B) For GOx three distinct regions with different pH dependency of the redox potential were observed. Slopes of -36 mV · pH-1 (green line), -57 mV · pH-1 (red solid line), and -27 mV · pH-1 (blue line) were calculated


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between pH 4.5 and pH 6.0, between pH 6.0 and pH 7.0, and between pH 7.6 and pH 8.5, respectively (values were determined in the presence of 110 mM Cl-). Below pH 6.0, the GOx redox potential was influenced by the presence of halides. Therefore at individual pH values additional measurements were carried out in Cl- free buffer solution (red dots).

By least squares fit of the FAD redox potentials in the pH ranges 4.0 – 6.5 and 7.4 – 8.8 slopes of -50 mV · pH-1 and -27 mV · pH-1 were calculated, respectively. The deviation from the theoretically expected values of -59 mV · pH-1 and -30 mV · pH-1 is within the experimental error. From the intersection of both straight lines (depicted in Figure 4A) a pKa of about 6.8 was calculated for the hydroquinone of free FAD, which is in agreement with previously published values.34,35 Interestingly, the pH dependent measurements of the GOx redox potential (depicted in Figure 4B) exhibited three distinct regions. From pH 6.0 to 7.0 a slope of -57 mV · pH-1 was obtained by least squares fit, which corresponds to a redox reaction following an ecec mechanism, in which two electrons and two protons are involved (Flox + 2e- + 2H+ ⇌ FlredH2). At basic pH (between pH 7.6 and 8.5) a slope of -27 mV · pH-1 was obtained, which corresponds to an ece mechanism, leading to the anionic (deprotonated) flavin hydroquinone species (Flox + 2e+ H+ ⇌ FlredH-). From the intersection of both straight lines a pKa of 7.2 was calculated for the flavohydroquinone of GOx. A minor shift in the pKa of the FAD hydroquinone by changing from the free to the enzyme bound state can be explained by the influence of the apoenzyme. Similar observations have been made previously for different types of flavodoxins, which show pKa values between 5.8 and 6.7 (in flavodoxins the flavin cofactor is FMN).42 The pKa value of 7.2 found in the current work for the flavohydroquinone is similar to the value of 7.4 which was


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calculated by Weibel and Bright during their kinetic investigation of the oxidative half-reaction in GOx.43 However, based on their NMR measurements of reduced GOx, Sanner and colleagues published a pKa value lower than 5.6.44

In the region between pH 4.5 and 6 for the GOx redox potential a pH dependency of only -36 mV · pH-1 was found. This behavior can be assigned to the influence of the chloride ion concentration on the GOx redox potential below pH 6.0. Below pH 6 an amino acid in close vicinity to the GOx bound FAD can be protonated, and this protonated state is stabilized in the presence of halides, especially Cl- (for details see supporting information). The reduction of protonated GOx follows an eec mechanism according to the following equation: ([GOx-Flox]H+ + 2e- + H+ ⇌ GOx-FlredH2). In order to prove that the rather low pH dependency of -36 mV · pH-1 between pH 4.5 and 6 is unequivocally caused by the presence of Cl-, we have carried out additional measurements in the absence of Cl- resulting in a larger slope, which is more close to the theoretically expected value of -59 mV · pH-1 expected for an ecec mechanism (for details see Figure 4B and supporting information). Both half reactions of GOx (glucose oxidation and dioxygen reduction) require the transfer of two electrons and two protons. As a consequence the overall reaction of GOx should reach its maximum rate below pH 7.2 when the GOx redox reaction follows an ecec mechanism. Indeed the highest catalytic activity of GOx has been observed between pH 4.0 and pH 7.0 with an optimum around pH 5.5).24,25 During their kinetic investigations of GOx, Weibel and Bright revealed that above pH 7.4 there was a considerable decrease of the overall turnover rate. They suggested that when the reduced form of the enzyme is present as flavohydroquinone anion, the oxidative half reaction (enzyme oxidation with concomitant dioxygen reduction) leads to the


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oxidized enzyme in a deprotonated state [GOx-Flox]-, which first needs to be protonated before it can enter the next catalytic cycle. Above pH 7 this protonation becomes the rate limiting step of the overall enzymatic reaction.43 For the reductive half reaction of GOx (with concomitant glucose oxidation) two different mechanisms involving an inner sphere complex or hydrid transfer have been discussed,43,45 whereof the latter seems to be more probable.45 According to Weibel and Bright the reductive half reaction is rate limiting at low pH in the presence and probably also in the absence of halides.43


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Table 1. Redox potentials including standard deviations of GOx and FAD in the pH range between pH 4.5 and pH 8.5, or pH 4.0 and pH 8.8, respectively. At each pH at least three measurements were carried out for samples containing chloride and one measurement for samples without chloride (values in brackets).

GOx

FAD

pH 4.0

n.d.

-47 ± 5

pH 4.5

30 ± 6 (46)

-81 ± 2

pH 5.0

13 ± 2 (25)

-105 ± 3

pH 5.5

-6 ± 1 (5)

-133 ± 9

pH 6.0

-23 ± 4

-156 ± 2

pH 6.5

-51 ± 7

-179 ± 2

pH 7.0

-80 ± 4

-207 ± 7

pH 7.4

-97 ± 3 (-97)

-212 ± 1

pH 7.6

-99 ± 6

-223 ± 4

pH 7.8

-109 ± 4

-228 ± 5

pH 8.0

-109 ± 7

-230 ± 8

pH 8.5

-126 ± 7

-241 ± 1

pH 8.8

n.d.

-243 ± 6

Conclusions In the current work UV/Vis spectroelectrochemistry has been used as a powerful method in order to determine the pH dependent redox potential and to elucidate mechanistic details of the redox chemistry of flavoenzymes by investigating the flavin model enzyme GOx. Between


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pH 4.5 and pH 8.5 the reduction of GOx leads directly to the formation of the (neutral or anionic) hydroquinone without formation of stable singly reduced semiquinone intermediates, if electrochemical equilibrium is established. During the spectroelectrochemical investigations the proper choice of the redox mediator mixture turned out to be a crucial parameter. If suitable mediators required for electrochemical equilibrium are missing, singly reduced GOx semiquinone radical species can be kinetically stabilized.21 As expected, the overall GOx redox potential was shifted to negative values with increasing pH. From the pH dependent measurements of the GOx redox potential a pKa of 7.2 has been determined for the GOx flavohydroquinone. In the range between pH 6 and pH 8.5 the GOx redox chemistry follows an ece(c) mechanism whereupon the redox potential of the first reduction is more negative than that of the second. At pH values ≤ pH 6.0 a dependency of the reduction mechanism of GOx and the GOx redox potential on the presence of halides, especially on Cl-, was observed. In the absence of Cl- the GOx redox potential follows an ecec mechanism and the pH dependency of the redox potential follows the general trend, which has been observed between pH 6.0 and pH 7.0. When in contrast Cl- ions are present, the active center of GOx, but not the redox active FAD as such, becomes protonated ([GOx-Flox]H+), and this protonated state becomes more stable at low pH. Thus, below pH 6.0 the reduction of GOx with protonated active center follows an eec mechanism according to the following equation: ([GOx-Flox]H+ + 2e- + H+ ⇌ GOx-FlredH2). Our results are in accordance with previous results, in which the catalytic activity of GOx at pH values below pH 6 was decreased in the presence of halides.43,46 Of main interest for the development of glucose biosensors and glucose biofuel cell anodes working at physiological or neutral pH are the GOx redox potentials at pH 7.4 and pH 7.0, which are independent from the


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presence or absence of halides. Here values of E1/2 pH 7.4 = -97 ± 3 mV and E1/2 pH 7.0 = -80 ± 4 mV have been determined.

ASSOCIATED CONTENT A detailed study of the influence of Cl- on the GOx redox potential at acidic pH, a comparison of the GOx redox potentials obtained by SVD and linearization, features of the spectroelectrochemical cell and the composition of the sample solutions are available as supporting information. This supplementary material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *[email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources This work was funded by the European Research Council (ERC) in the 7th EU Research Framework Program (FP7/2007-2013)/ ERC Grant agreement no. 240544 and the state North Rhine Westphalia. ACKNOWLEDGMENT


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We thank Mr. Bernd Meyer for the construction of the spectroelectrochemistry cell and AttoTec for measurements at the Lambda 750 photospectrometer. Further we thank Dr. Peter Haring Bolivar, Dr. Marcin Grzegorzek and Mr. Oliver Tiebe for kind support at the University of Siegen.

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BRIEFS: Spectroelectrochemical investigation of the redox enzyme glucose oxidase (GOx)

SYNOPSIS


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Determination of the pH Dependent Redox Potential of Glucose

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