bioelectrocatalysis by membrane-bound aldehyde dehydrogenase from Gluconobacter oxydans and cyanide effects on its bioelectrocatalytic properties

The bioelectrocatalytic properties of membrane-bound aldehyde dehydrogenase (AlDH) from Gluconobacter oxydans NBRC12528 were evaluated. AlDH exhibited direct electron transfer (DET)-type bioelectrocatalytic activity for acetaldehyde oxidation at several kinds of electrodes. The kinetic and thermodynamic parameters for bioelectrocatalytic acetaldehyde oxidation were estimated based on the partially random orientation model. Moreover, at the multi-walled carbon nanotube-modified electrode, the coordination of CN (cid:0) to AlDH switched the direction of the DET-type bioelectrocatalysis to acetate reduction under acidic conditions. These phenomena were discussed from a thermodynamic viewpoint.


Introduction
Acetic acid bacteria have a variety of membrane-bound dehydrogenases that work in their unique metabolism such as acetic acid fermentation [1].The application of these bacteria in bioelectrochemical devices (biosensors, biofuel cells, biosupercapacitors, etc.) is significant for industry [1].Specifically, membrane-bound dehydrogenases such as fructose dehydrogenase (FDH) [2], alcohol dehydrogenase (ADH) [3][4][5], gluconate dehydrogenase (GaDH) [6], and lactate dehydrogenase (LDH) [7] are known to directly communicate with electrodes and proceed electro-enzymatic reactions, which is called direct electron transfer (DET)-type bioelectrocatalysis.It is suggested that these dehydrogenases transfer electrons from their substrates to the electrodes via their catalytic centers and one or more hemes c in this order [1].
Herein, we focus on another membrane-bound dehydrogenase of acetic acid bacteria, namely, aldehyde dehydrogenase (AlDH).AlDH from Gluconobacter oxydans NBRC12528 is a heterotrimeric enzyme composed of a large subunit (86 kDa) containing a molybdopterin cofactor (Moco) as a catalytic center, a membrane-bound cytochrome c subunit (55 kDa) containing three hemes c, and a small subunit (the molecular mass of which is unknown) containing an iron-sulfur cluster [8,9].In vivo, AlDH oxidizes acetaldehyde, and the extracted electrons are transferred to ubiquinone in the inner membrane [9].However, the electrochemical properties and the bioelectrocatalytic performance of AlDH have not yet been examined.
In the present study, we report DET-type bioelectrocatalysis by AlDH at planar gold (Au), 2-mercaptoethanol (ME)-functionalized Au, and multi-walled carbon nanotube (MWCNT)-modified glassy carbon (GC) electrodes.Planar and ME-functionalized Au electrodes were used as platforms for DET-type bioelectrocatalysis by FDH [2,10].MWCNTs are nanostructured electrode materials that promote various DET-type reactions [11] with the following effects: 1) the enlarged effective surface area of the electrode increases the amount of adsorbed enzymes [12]; 2) the curvature of the mesoporous electrode structure increases the probability of enzyme orientations suitable for DET-type reactions [13,14]; 3) the electric field strengthened by the expansion of the electric double layer accelerates the kinetics of heterogeneous electron transfer at the edge of the micropores [15].
The effects of the coordination of cyanide ions (CN − ) to the hemes c on the bioelectrocatalytic properties of AlDH are also evaluated herein.Interestingly, CN − -coordinated AlDH exhibits DET-type activity for acetate reduction.

Materials and chemicals
Protein markers for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were obtained from Nacalai Tesque Inc. (Japan).Water-dispersed MWCNTs were kindly donated by Nitta Co. (Japan).Other reagents were purchased from Wako Pure Chemical Industries, Ltd. (Japan).All solutions were prepared using ultrapure water.

Purification of AlDH
AlDH was purified from a variant of Gluconobacter oxydans NBRC12528 (ΔadhA::Km r [16]) according to the procedure in the literature [8], with minor modifications.Briefly, the process of removing ADH from the membrane fraction using multiple surfactants was omitted because the gene of ADH was knocked out.The purified enzyme solution contained 0.5% Triton® X-100 as a solubilizer and 25 mM benzaldehyde and 10% sucrose as stabilizers.The results of SDS-PAGE analysis are shown in Fig. S1, which demonstrate that the subunit structure of AlDH from the variant was identical to the reported structure of that purified from the wild-type cells [8].
The AlDH activity was spectrophotometrically measured using potassium ferricyanide as an electron acceptor and ferric sulfate-Dupanol reagent [8].Here, one unit (U) of AlDH activity is defined as the amount of the enzyme that oxidizes 1 μmol of acetaldehyde per minute at pH 4.0.The protein concentration was estimated using a DC protein assay kit (Bio-Rad, USA) with bovine serum albumin as the standard.The specific activity (approximately 600 U mg − 1 ) was slightly better than that described in the literature (430 U mg − 1 [8]), plausibly due to the shortening of the purification process, as described above.

Electrode preparation
Au and GC electrodes (3 mm in diameter, BAS, Japan) were polished with 1.0 μm and 0.05 μm alumina powder.The electrodes were subsequently rinsed by sonication in distilled water.Self-assembled monolayers of 2-mercaptoethanol (ME) were then formed on the Au electrodes by immersing the Au electrodes in an ethanol solution containing 2 mM ME for 2 h.Subsequently, the ME-functionalized Au electrodes were washed with ethanol and distilled water.MWCNTmodified GC electrodes were also prepared by applying 60 μL of 0.1 wt% MWCNT dispersion to the surface of each GC electrode, followed by drying at 70 • C.

Electrochemical measurements
All electrochemical measurements were performed at 25 • C with electrochemical analyzers (CV-50 W (BAS, USA) or ALS 701E (ALS Co. Ltd., Japan)).Anaerobic measurements were carried out in a nitrogen (N 2 ) chamber filled with a mixture of 96% N 2 and 4% H 2 .A platinum wire and a homemade Ag|AgCl|sat.KCl electrode were used as the counter and reference electrodes, respectively.In this study, all potentials are referred to the reference electrode.adding AlDH at a final concentration of 2 μg mL − 1 into the electrolysis solution, clear sigmoidal waves were observed at both electrodes (Fig. 1A).As shown in Fig. S2, the anodic current density gradually increased with time and finally reached the maximum value.Thus, these waves are ascribed to DET-type bioelectrocatalysis of acetaldehyde oxidation by AlDH physically adsorbed on the electrodes.The bioelectrocatalytic current density reached approximately 20 μA cm − 2 at the planar Au electrode and 30 μA cm − 2 at the ME-functionalized Au electrode at 0.4 V. On the other hand, the slightest anodic current was also observed in the absence of acetaldehyde (Fig. S3), which seems to be due to the DET-type oxidation of benzaldehyde contained in an enzyme solution.

DET-type bioelectrocatalytic acetaldehyde oxidation by AlDH
In this study, kinetic and thermodynamic analysis of the DET-type bioelectrocatalytic waves at the AlDH-modified electrodes was performed using a partially random orientation model in which it was assumed that the enzymes were adsorbed on the electrode surface in a homogeneously distributed orientation [17,18].In the model, the steady-state current density (j) is expressed by the following equation [17]: where j cat is the limited steady-state catalytic current density, k • max is the standard rate constant for heterogeneous electron transfer between the electrode and the redox center of AlDH in the best orientation, k c is the catalytic constant, Δd is the difference in the distance between the closest and farthest approaches of the redox center of AlDH that electrochemically communicates with an electrode, α is the transfer coefficient (assumed to be 0.5 in this case), and β is the decay coefficient of long-range electron transfer (assumed to be 1.4 Å − 1 for proteins [19]).In addition, j cat and η are given as follows: where n S is the number of electrons in acetaldehyde oxidation (=2), n ′ E is the number of electrons in the rate-determining step of the heterogeneous electron transfer (=1 in general), Γ E,eff is the surface concentration of the effective enzyme immobilized on the electrode, E is the electrode potential, E •′ E is the formal potential of the electrode-active redox center of AlDH, F is the Faraday constant, R is the gas constant, and T is the absolute temperature.Using E •′ E , k • max / k c , Δd, and j cat as adjustable parameters, Eq. ( 1) was fitted to the steady-state catalytic waves using non-linear regression analysis with Gnuplot.The background currents were subtracted from the total currents before analysis.The fitting results are shown in Fig. 1B, and the refined numerical data are summarized in Table 1.The errors are due to the variation in the characteristics of enzyme-modified electrodes.
The E •′ E values seem to be assigned to the electrode-active heme c, as compared to the spectrophotometrically determined values of the formal potentials of hemes c in FDH (E •′ = 150 mV, 60 mV, and − 10 mV) [20].The value of E •′ E appeared to be independent of the surface modification of the Au electrode.In addition, there was no significant difference in the estimated Δd values.On the other hand, the values of k • max / k c and j cat at the ME-functionalized Au electrode were larger than those obtained with the planar Au electrode.When it is assumed that the k c value is equal at both electrodes, it can be concluded that k • max and Γ E,eff increase due to modification of the Au electrodes with ME.In the case of FDH, the electrode modification affects not Δd but Γ E,eff [21].The similar improvement in the DET-type bioelectrocatalysis by ME modification was explained by applying a surfactant bilayer model [10].Briefly, the hydrophilic surface of the ME-functionalized electrode is favorable for forming the Triton® X-100 bilayer into which the enzymes are embedded, which increases Γ E,eff and shortens the distance between the electrode-active center of the enzyme and the electrode.Similar effects are expected in this case.However, further studies are required for in-depth discussion of how surface modification of the electrode affects the DET-type bioelectrocatalysis by AlDH.

Effects of CN − on reverse DET-type bioelectrocatalysis by AlDH
We investigated the effects of CN -on DET-type bioelectrocatalysis by AlDH.CN − is known to be coordinated to the axial ligand of the heme iron, which results in a shift in the redox potential by approximately 0.4 V toward the negative direction [22].After the addition of 1 mM potassium cyanide (KCN), a small bioelectrocatalytic wave of acetaldehyde oxidation at the AlDH-modified Au electrode was first observed, but the intensity of the wave gradually decreased with time and the signal finally disappeared (Fig. S4).The results can be explained by the following mechanisms: 1) The coordination of CN − to the hemes c in AlDH and the adsorption of AlDH on an electrode proceed simultaneously, where the former is slower than the latter because the dissociation of a proton from HCN (pK a = 9.2) is unfavorable under acidic conditions.(Caution: addition of KCN to acidic solutions must be performed with good ventilation as the process generates HCN).
2) Intramolecular electron transfer in AlDH is inhibited by CN − coordination.This means that the electrons are transferred from the catalytic center to the electrode through one or more hemes c in the native enzyme.
3) The formal potential of the Moco (E •′ Moco ) in AlDH is assumed to be close to that of xanthine oxidase (− 0.5 V [24]), which is a typical molybdenum enzyme.The involvement of other electron-mediating redox centers was ignored here.The downhill property is well explained by electron transfer from acetaldehyde to the electrode-active heme c at pH 4.0.However, the CN − coordination to the heme c results in an uphill barrier in the intramolecular electron transfer process, which inhibits the enzyme activity for acetaldehyde oxidation (Fig. 2A).
At lower pH (pH 2.5), E •′ S shifts toward positive potential and becomes more positive than E • Moco .Therefore, it was expected that electron transfer from the CN − -coordinated heme c to acetate via Moco would be thermodynamically favorable, as shown in Fig. 2B.This hypothesis was tested by using a MWCNT-modified GC electrode as a platform for AlDH because the electrode provided a relatively smaller background current probably ascribed to proton reduction under strongly acidic conditions, compared to the planar Au and GC electrodes.In addition, DET-type bioelectrocatalytic acetaldehyde oxidation was observed at the AlDH-adsorbed MWCNT-modified GC electrodes, with a larger current density than that observed at AlDH-modified Au electrodes (Fig. S5).Since the AlDH solution at pH 2.5 kept the activity for acetaldehyde oxidation, there was no fatal change in the enzyme conformation under acidic conditions.Fig. 3 shows the CVs recorded at the MWCNT-modified GC electrodes in an McB (pH 2.5) containing 0.1 M acetic acid and 1 mM KCN under anaerobic conditions.After addition of AlDH at a final concentration of 2 μg mL − 1 into the electrolysis solution, the cathodic DET-type bioelectrocatalytic current for acetate reduction by CN − -coordinated AlDH was observed.An increase in the current was only observed in the presence of AlDH, KCN, and acetate.Here, it is noteworthy that the current density increased over many hours, as demonstrated by the multi-scanned CVs in Fig. S6.The current decrease in the first 2 h is plausibly due to adsorption of AlDH on the electrodes, and CN − was gradually coordinated to AlDH, which seemed to proceed very slowly, as described above.Only the current decrease was observed in the absence of KCN (Fig. S7), which also supports the above process.In addition, the onset potential (approximately − 0.6 V) is somewhat more negative than that expected from the formal potential of the CN − -coordinated electrode-active heme c.The potential shift induced by CN − coordination might be larger than 0.4 V.However, DET-type acetate reduction cannot be observed in the presence of high concentrations (~mM) of acetaldehyde (data not shown), which is suggested to be due to the biased catalytic property and the product inhibition.

Table 1
Refined parameters from non-linear regression analysis of voltammograms.The errors were evaluated from Student's t-distribution at a 90% confidence level (n = 5).T. Adachi et al.
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Fig. 1
Fig. 1 shows cyclic voltammograms (CVs) recorded at planar and ME-functionalized Au electrodes in a McIlvaine buffer (McB) (pH 4.0) containing 0.1 M acetaldehyde under Ar atmospheric conditions.After

Fig. 1 .
Fig. 1. (A) CVs for acetaldehyde oxidation at the AlDH-modified planar Au electrode (broken black line) and ME-functionalized Au electrode (solid red line) in an McB (pH 4.0) containing 0.1 M acetaldehyde and 2 μg mL − 1 AlDH at 25 • C under quiescent and Ar atmospheric conditions, at a scan rate (v) of 10 mV s − 1 .CVs in the absence of AlDH are represented by dotted lines.(B) Background-subtracted linear sweep voltammogram for acetaldehyde oxidation at the AlDH-modified planar Au electrode (black circles) and ME-functionalized Au electrode (red squares) in an McB (pH 4.0) containing 0.1 M acetaldehyde and 2 μg mL − 1 AlDH at 25 • C under quiescent and Ar atmospheric conditions, at v = 10 mV s − 1 .The dotted and broken lines indicate the refined curves estimated by non-linear regression analysis based on Eq. (1).(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2
presents the schematics of the electron transfer pathway of AlDH.The approximated values of the formal potential at pH 4.0 were evaluated based on the following considerations: a) the E •′ E value of one of hemes c obtained in this work, b) the formal potential of the acetate/ acetaldehyde redox couple (E •′ S ) is − 0.78 V at pH 7.0 [23] and shifts by − 89 mV pH − 1 based on the following and the Nernst equations:

Fig. 2 .
Fig. 2. Proposed potential profiles for acetaldehyde oxidation at pH 4.0 (A) and acetate reduction at pH 2.5 (B) in DET-type bioelectrocatalysis by AlDH.Heme c E indicates the electrode-active heme c.

Fig. 3 .
Fig. 3. CVs for acetate reduction at the AlDH-adsorbed MWCNT-modified GC electrode in an McB (pH 2.5) containing 0.1 M acetic acid and 1 mM KCN in the presence (solid line) and absence (dotted line) of 2 μg mL − 1 AlDH at 25 • C under quiescent and anaerobic conditions, at v = 10 mV s − 1 .
Self-archived copy in Kyoto University Research Information Repository https://repository.kulib.kyoto-u.ac.jp T. Adachi et al.A