Accepted Manuscript

Title: Low Potential Catalytic Voltammetry of Human SulfiteOxidase

Author: Palraj Kalimuthu Abdel A. Belaidi Guenter SchwarzPaul V. Bernhardt

PII: S0013-4686(16)30180-3DOI: http://dx.doi.org/doi:10.1016/j.electacta.2016.01.181Reference: EA 26563

To appear in: Electrochimica Acta

Received date: 10-12-2015Revised date: 21-1-2016Accepted date: 24-1-2016

Please cite this article as: Palraj Kalimuthu, Abdel A.Belaidi, Guenter Schwarz,Paul V.Bernhardt, Low Potential Catalytic Voltammetry of Human Sulfite Oxidase,Electrochimica Acta http://dx.doi.org/10.1016/j.electacta.2016.01.181

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1

Low Potential Catalytic Voltammetry of Human Sulfite Oxidase

Palraj Kalimuthu,a Abdel A. Belaidi,b Guenter Schwarzb and Paul V. Bernhardta,*

aSchool of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, 4072, Australia

b Institute of Biochemistry, Department of Chemistry & Center for Molecular Medicine, Cologne University, Zülicher Str. 47, 50674 Köln, Germany

E-mail: p.bernhardt@uq.edu.au

2

–200 0 200

0

1

/ mV vs. NHEE

I

a

b

Graphical Abstract

Highlights

Human sulfite oxidase (HSO) catalyses the oxidation of sulfite to sulfate.

Fe(III) hexa-amine complexes may act as synthetic electron acceptors from HSO.

Electrochemical sulfite oxidation can be achieved with HSO and the Fe(III) complexes.

Varying the Fe(III/II) redox potential of the mediators results in different voltammetry.

A sulfite biosensor is constructed and used to determine sulfite in wine and beer samples.

3

Abstract

Mediated electrocatalytic voltammetry of human sulfite oxidase (HSO) is demonstrated with synthetic

one electron transfer iron complexes bis(1,4,7-triazacyclononane)iron(III) ([Fe(tacn)2]3+) and 1,2-

bis(1,4,7-triaza-1-cyclononyl)ethane iron(III) ([Fe(dtne)]3+) at a glassy carbon working electrode. The two

electron acceptors for HSO, differing in redox potential by 270 mV, deliver different driving forces for

electrocatalysis. Digital simulation of the catalytic voltammetry was achieved with single set of enzyme-

dependent kinetic parameters that reproduced the experimental data across a range of sweep rates,

and sulfite and mediator concentrations. Amperometry carried out in a stirred solution with the lower

potential mediator [Fe(tacn)2]3+ was optimised and exhibited a linear increase in steady state current in

the sulfite concentration range from 5.0 × 10-6 to 8.0 × 10-4 M with a detection limit of 0.2 pM (S/N = 3).

The HSO coupled electrode was successfully used for the determination of sulfite concentration in white

wine and beer samples and the results validated with a standard spectrophotometric method.

Keywords: enzyme; molybdenum; sulfite

1. Introduction

The molybdenum-dependent sulfite oxidizing enzymes comprise sulfite oxidase (SO) and sulfite

dehydrogenase (SDH) [1, 2]. SO is found in animals and plants whereas SDH is only found in bacteria [3].

Only the plant SO is a true oxidase while all other sulfite oxidizing enzymes donate electrons to

cytochrome c. Vertebrate SOs can use either cytochrome c or dioxygen as an electron acceptor. Only

4

one crystal structure is available for a vertebrate SO (from chicken liver) [4] revealing a 103 kDa

homodimer in which each subunit contains a negatively charged small heme b domain at the N-terminus

and positively charged larger molybdopterin domain at the C-terminus. The heme accepts electrons

from the Mo ion following sulfite oxidation. A flexible connects the Mo and heme domains which are

more than 30 Å apart in the crystal structure conformation; a distance too great for electron transfer.

Spectroscopic and kinetic studies have demonstrated that the heme b domain swings around to be in

proximity to the molybdenum active site where electron transfer (Mo to heme) can take place after

sulfite oxidation [5-7].

Human sulfite oxidase (HSO) shares a 68% sequence identity with chicken SO [4]. Among the

eukaryotic SOs, HSO has been studied extensively because of its role in the potentially fatal disease SO

deficiency [8, 9]. The physiological role of SO is to remove toxic sulfite (a product of organo-sulfur

compound metabolism) and covert it to chemically inert sulfate. Despite its name the physiological

electron acceptor of SO is in fact cytochrome c (eq. 1). In the catalytic reaction, SO is active in its fully

oxidized state (MoVI) in which molybdenum is coordinated by a cysteine thiolate, the dithiolene group of

molybdopterin, and two terminal oxygen atoms as shown in Scheme 1 [7, 10-12]. Upon reaction with

sulfite, one oxido ligand is transferred to sulfite to give sulfate and the Mo ion is reduced its tetravalent

state. Subsequently, hydroxide displaces sulfate, and the removal of this hydroxido ligand proton occurs

spontaneously when the Mo ion is reoxidised to its hexavalent state by two cytochrome c molecules.

There have been a number of electrochemical investigations of SO and SDH enzymes from

different organisms. In these cases the electrode is the ultimate electron acceptor resulting in an anodic

catalytic current. Electrons may be transferred directly from the enzyme [2, 13-17] or via a mediator

which may be synthetic [18-20] or natural (cytochrome c) [18, 21-25].

5

The dynamics HSO are potentially problematic for efficient electrocatalysis. While the Mo and

heme cofactors are separated, the enzyme is unable to be reactivated through reoxidation. It is of

interest whether confinement of HSO enzyme to a thin layer at the electrode surface suppresses this

motion. Spectroelectrochemistry of HSO showed the FeIII/II redox potential to be +62 mV vs NHE (pH 7.5)

[6]. At applied electrochemical potentials above this value, the enzyme will be continually reoxidsed and

reactivated for sulfite oxidation. To achieve this we employed two artificial electron acceptors; the hexa-

amine complexes [Fe(tacn)2]3+ and [Fe(dtne)]3+ (Fig. 1) with redox potentials of +144 and +415 mV vs

NHE, respectively which present significantly different overpotentials but are structurally almost the

same. The higher FeIII/II redox potential of [Fe(dtne)]3+ is due to the presence of two tertiary amines

compared to the all-secondary amine [Fe(tacn)2]3+. It is notable that nonspecific oxidation of sulfite at an

electrode (without any enzyme present) is inevitable above ca. +550 mV vs NHE [26] and this places an

upper bound on the redox potential of any mediator in a sulfite oxidizing electrochemical system.

An additional feature of this study is electrochemical simulation of the experimental

voltammetry. Given that the catalytic cycle involves several steps, some chemical reactions between

HSO and sulfite/sulfate and some being outer sphere electron transfer reactions between HSO and the

mediators, a set of rate constants can be defined (Scheme 1). These rate constants must be able to

reproduce the catalytic voltammetry under a variety of conditions including sweep rate, mediator

concentration and sulfite concentration. Finally, amperometry is employed to estimate the lowest

detection limit and linear current response for the determination of sulfite in aqueous solution and in

the quantification of sulfite in beer and wine samples where it is a commonly found as an additive to

combat spoilage from oxidation and microbial activity [27, 28].

6

2. Experimental

2.1 Materials

Human sulfite oxidase (HSO) was purified in E. coli TP1000 as previously described [29]. The iron

complexes bis(1,4,7-triazacyclononane)iron(III) bromide ([Fe(tacn)2]Br3) [30] and 1,2-bis(1,4,7-triaza-1-

cyclononyl)ethane iron(III) bromide ([Fe(dtne)]Br3.3H2O) [31] were synthesized according to the

previous procedures. Sodium sulfite and 5,5’-dithio-bis(2-nitrobenzoic acid) (Ellman’s reagent) were

purchased from Aldrich and were used as received. The beer and wine samples were purchased from

local retail outlets. All other reagents used were of analytical grade purity and used without any further

purification. Tris acetate buffer (50 mM) was used for all experiments at pH 8.0. For pH-dependent

experiments, the mixture of buffers (20 mM MES buffer pH 5.5–6.7, 20 mM Bis-Tris buffer pH 5.8–7.2,

20 mM Tris buffer pH 7.0–9.0, 20 mM CHES buffer pH 8.6–10.0 and 20 mM CAPS buffer pH 9.7-11.1) was

used and the desired pH was obtained with dilute acetic acid or NaOH. All solutions were prepared with

ultrapure water (resistivity 18.2 MΩ.cm) from a Millipore Milli-Q system.

2.2 Electrochemical Measurements and Electrode Cleaning

The cyclic voltammetry (CV) and chronoamperometry experiments were carried out with a BAS

100B/W electrochemical workstation. A three-electrode system was employed comprising a glassy

carbon (GC) disk working electrode, a Pt wire counter, and an Ag/AgCl reference electrode (+196 mV vs

NHE). Potentials are cited versus NHE. Experiments were carried on solutions that had been purged with

argon gas for 30 min. The GC electrode was polished with 0.50 and 0.05 μm alumina slurry and then

rinsed thoroughly with water. Then the electrode was sonicated in water for 10 min to remove adsorbed

alumina particles and dried in a nitrogen atmosphere.

7

The electro-active surface area of the GC electrode (A) was determined from the cyclic

voltammetry of 1 mM ferrocene methanol [32] in 0.1 M KCl solution at different sweep rates using the

Randles-Sevcik equation (equation 1) [33].

ip = (2.69 × 105)n3/2ADo1/2Coν1/2 (1)

The standard diffusion coefficient (Do) of ferrocene methanol is 6.7 × 10-6 cm2 s-1 [34], ip is the

measured current maximum, n is the number of electrons, Co is concentration of analyte (mol cm-3), and

ν is the sweep rate (V s-1).

The variation of the catalytic current (ilim) as a function of sulfite concentration was fit to

Michaelis–Menten kinetics (equation 2) yielding KM,app (the apparent Michaelis constant) and imax (the

effective electrochemical turnover number, imax = nFA[HSO]) [35].

𝑖lim =𝑖max[SO3

2−]

𝐾M,app + [SO32−]

(2)

The pH dependence of the catalytic current was modeled by equation 3 [11] which is applicable

for an active form of the enzyme that is deactivated by either deprotonation of an acid at high pH (pKa1)

or protonation of a base at lower pH (pKa2).

𝒊𝐦𝐚𝐱(𝐩𝐇) =𝒊𝐨𝐩𝐭

𝟏 + 𝟏𝟎(𝐩𝐇−𝐩𝑲𝐚𝟏) + 𝟏𝟎(𝐩𝑲𝐚𝟐−𝐩𝐇) (𝟑)

2.3 Enzyme Electrode Preparation

A 3 µL droplet of HSO (66 µM) in 50 mM Tris buffer (pH 8.0) was pipetted onto the conducting

surface of an inverted, freshly prepared GC working electrode and this was allowed to dry to a film at

4°C. To prevent protein loss the electrode surface was carefully covered with a semi-permeable dialysis

membrane (SERVA MEMBRA-CEL, molecular weight cut off 3500 Da), presoaked in water. The dialysis

membrane was pressed onto the electrode with a Teflon cap and fastened to the electrode with a

rubber O-ring to prevent leakage of the internal membrane solution. The resulting modified electrode

8

was stored at 4°C in 50 mM Tris buffer (pH 8.0) when not in use. The enzyme was confined to a thin

layer beneath the membrane while substrate and mediators were able to diffuse across the dialysis

membrane.

2.4 Electrochemical Simulation

The DigiSim program (version 3.03b) was employed to simulate the experimental cyclic

voltammograms [36]. The experimental parameters restrained in each case were the working electrode

surface area (0.055 cm2) and the double-layer capacitance (12 µF). Semi-infinite diffusion was assumed

and all pre-equilibration reactions were enabled. The apparent redox potential of mediators was

determined from control experiments in the absence of enzyme or substrate. The diffusion coefficients

of mediators were also obtained in the presence of a dialysis membrane covering the electrode by

simulation of the cyclic voltammetry at different sweep rates in the absence of substrate and enzyme to

give value of 5 ×10-6 cm2 s-1. The diffusion coefficients for HSO and substrate were taken to be 5 ×10-7

and 5 ×10-6 cm2 s-1 [26]. These values were kept constant for simulating the various substrate- and

mediator-concentration-dependent CVs. The heterogeneous rate constant (k0) was determined from

simulating the sweep rate dependence of the anodic peak to cathodic peak separation of mediators (in

the absence of HSO) and then held constant thereafter. The only values that were allowed to differ were

the rate constants for the outer sphere electron transfer reaction between each mediator and enzyme

(k4, k4’, k-4 and k-4’ in Scheme 2). It was assumed that k4 = k4’ and k-4 = k-4’ i.e. oxidation of either the

MoV/FeII or MoVI/FeII forms of HSO proceeded at the same rate, which is reasonable given that the heme

cofactor is the site of oxidation and its redox potential is known (+62 mV vs NHE) [6].

9

2.5 Spectrophotometric Sulfite Determination

As a complement to amperometric sulfite determination the results were validated using

Ellman’s reagent which is cleaved by sulfite to form an organic thiosulfate and 5-mercapto-2-nitroben-

zoate stoichiometrically; the latter being determined spectrophotometrically [37].

3. Results and Discussion

3.1 Electrocatalytic Mechanism of HSO

The electrocatalytic mechanism of HSO is illustrated in Scheme 2. The single electron transfer

acceptors [Fe(tacn)2]3+ and [Fe(dtne)]3+ used in the present study are synthetic substitutes for

cytochrome c and so two consecutive one electron transfer reactions are necessary to regenerate the

reduced HSO to its active form after it has been reduced by sulfite.

There are also two intramolecular electron transfer (IET) processes. The first IET step occurs

when MoIV transfers one electron to the oxidized ferric heme b cofactor. The ensuing MoV/FeII species

transfers an electron to the artificial electron acceptor producing the MoV/FeIII state (rate constant k4). A

second IET step generates the MoVI/FeII state, and reduction of a second molecule of mediator (rate

constant k4’) regenerates the fully oxidized MoVI/FeIII state of the enzyme. The sum of the forward and

reverse IET2 steps is known (ket > 400 s-1 at pH 7) [38] and this first order reaction is always much faster

than the rates of the (second order) outer sphere redox reactions (k4, k4’) which are slowed down by the

low concentrations of mediators and HSO used in this experiment. For this reason we have not included

either IET step in our kinetic model i.e. it is assumed to be fast and never rate limiting.

10

The substrate (sulfite) and mediator ([Fe(tacn)2]3+ or ([Fe(dtne)]3+) are under diffusion control

while HSO is confined to the small volume under the membrane but still may diffuse within that space.

We have assumed that the catalytic reaction follows Michaelis-Menten kinetics comprising substrate

binding (k1/k-1), turnover (k2/k-2) and product release (k3/k-3). A simplified double substrate ‘ping-pong’

mechanism is appropriate for this type of catalysis.

3.2 Mediator Voltammetry

Interestingly upon introduction of 10 µM of the mediator [Fe(tacn)2]3+ into the electrochemical

cell no significant redox response was observed initially (in the first cycle) at the dialysis membrane

covered HSO modified GC electrode (Supporting Information, Figure S1A). In the second cycle the redox

response of [Fe(tacn)2]3+ emerged and increased in current up to about 12 cycles where a consistent

waveform was established.

The GC/HSO electrode shows a well-defined redox wave centred at ca. +56 mV vs NHE for 10

µM [Fe(tacn)2]3+ with a peak to peak separation of only 42 mV in 50 mM Tris buffer solution (Figure 2,

curve a). There are several important features to note. The increasing current at the GC/HSO electrode

as a function of cycle number (Supporting Information, Figure S1A) indicates that the [Fe(tacn)2]3+

molecules only cross the membrane slowly from the bulk solution i.e. flux across the membrane during

the sweep is insufficient to keep pace with depletion of the mediator from the diffusion layer in the

initial sweep. Moreover, the establishment of a consistent CV after several cycles indicates that the

amount of [Fe(tacn)2]3+ that eventually accumulates under the membrane is sufficient to sustain

catalysis i.e. it is not depleted during the sweep (see below). Secondly the peak to peak separation is less

than 57 mV but greater than 0 mV, which is intermediate of a response governed by linear diffusion and

that seen in a thin layer cell [33]. Furthermore the symmetry of the wave is a hybrid of the tailing

11

waveform characteristic of normal linear diffusion and the symmetrical wave characteristic of a thin

layer cell due to the confines of the membrane [33]. It is also apparent that the observed currents are

much greater than would be expected for a 10 µM solution on the basis of equation 1. It appears that a

significant amount of [Fe(tacn)2]3+ is concentrated on the inner side of the dialysis membrane and

escape to the bulk solution (10 µM) is slow. If the electrode is transferred to a buffer solution containing

no Fe complex then the current response is significantly diminished upon continuous cycling (Supporting

information Figure S1B). On the basis of the maximum peak height attained and equation 1 we have

estimated the amount maximum concentration of [Fe(tacn)2]3+ under the dialysis membrane to be ca.

120 µM. The redox response was investigated at different scan rates. Both oxidation and reduction

currents increase with scan rate from 10 to 100 mV s-1 (Supporting information Figure S2A) but the

linear increase of peak height with the square root of sweep rate (R2 = 0.999, with zero intercept) is

more consistent with a linear diffusion controlled process than a thin layer response (linear increase of

current with sweep rate). On balance the mediator redox response is predominantly under diffusion

control (Supporting information Figure S2B).

Also of interest is the deviation of the [Fe(tacn)2]3+/2+ redox potential under these conditions (E’

+56 mV vs NHE) from that obtained in solution without a membrane and in the absence of HSO (+144

mV). This is not a consequence of the membrane as the CVs of [Fe(tacn)2]3+ alone are the same in the

absence or presence of the membrane (Figures S1C and S1D). The pronounced cathodic shift in the

[Fe(tacn)2]3+/2+ redox potential is only seen in the presence of HSO and this is attributed to the formation

of a non-covalent (outer sphere) complex with HSO under the membrane. The natural electron acceptor

of HSO is the highly positively charged protein cytochrome c which is thought to bind (non-covalently) at

a negatively charged surface of HSO adjacent to the heme cofactor. The affinity of the tri-positively

12

charged [Fe(tacn)2]3+ evidently mimics cytochrome c and presumably interacts with the same highly

negatively charged surface of HSO and this interaction lowers the redox potential of the mediator.

The same analysis was carried out with [Fe(dtne)]3+ and once again the CV behaviour is

consistent with a predominantly diffusion controlled response this time at a much higher redox

potential (Supporting Information Figure S3). Again the observed [Fe(dtne)]3+/2+ redox potential (+355

mV) was shifted cathodically from its value in solution (+410 mV) [31].

3.3 Catalytic Voltammetry

Although HSO has two electroactive centers (Mo and heme) no redox responses were observed

from either cofactor in the absence (or presence) of sulfite at the GC electrode without mediator

present (data not shown). This is not unexpected as direct electrochemistry of HSO has only been

observed at chemically modified Au [39], Ag [17] or Sb-doped SnO2 [40] electrodes and only quite weak

responses were seen.

In the presence of HSO (under the membrane), [Fe(tacn)2]3+ and sulfite (5 mM), a well-defined

classic sigmoidal waveform is seen and the limiting anodic current increases by an order of magnitude

(Figure 2, curve b). No cathodic wave is present in this case and obviously the forward and backward

sweeps are same when the charging current is taken into account. In a control experiment, we found

that the redox response of [Fe(tacn)2]3+ is insensitive to 5 mM sulfite at a bare GC electrode (data not

shown) within the potential window of -200 to +350 mV vs NHE indicating that mediator alone cannot

oxidize sulfite. Thus, the observed sigmoidal form of voltammetry at enzyme modified electrode is

characteristic of a catalytic homogeneous reaction coupled to heterogeneous electron transfer (EC’

13

mechanism) [33] where sulfite is oxidized enzymatically yielding the reduced form of enzyme (MoIV),

which is reoxidized by electro-generated [Fe(tacn)2]3+.

3.4 HSO-Sulfite Reaction

The reaction between HSO and sulfite was investigated by varying the sulfite concentration

while maintaining a constant concentration of mediator and enzyme. The examples in Figure 3 show the

CVs of the GC/HSO electrode in the presence of 10 µM [Fe(tacn)2]3+ (Figure 3A) and 20 µM [Fe(dtne)]3+

(Figure 3B) at a sweep rate of 5 mV s-1 in Tris buffer (pH 8). In both cases, the CVs take the form of an

asymmetric transient catalytic wave up to 800 µM sulfite with a pronounced anodic peak but no

corresponding cathodic current. The peak is due to mass transport limitations where sulfite becomes

depleted at the electrode surface due to the rate it is consumed by HSO, which cannot be sustained by

diffusion from the bulk solution across the membrane. It is apparent that as the sulfite concentration is

increased further, (3.2 mM) the wave increases in magnitude and the sharp transient form of the wave

becomes more symmetrical. Ultimately (> 4 mM sulfite), the transient wave becomes sigmoidal where

the concentration of sulfite within the reaction layer is constant during the sweep. The sigmoidal

waveform is indicative of an electrochemical steady state i.e. the oxidized form of mediator is consumed

(by homogeneous reaction with HSOred) at the same rate that it is generated at the electrode surface

and mass transport of sulfite from the bulk is fast enough to ensure its concentration is constant under

the membrane. CVs at all concentrations of sulfite examined appear in the Supporting Information with

[Fe(tacn)2]3+ (Figure S4A) and [Fe(dtne)]3+ (Figure S5A) as mediator.

14

The catalytic sulfite oxidation current increased linearly up to 800 and 1600 µM sulfite before

saturating at millimolar concentrations. Apparent Michaels constants (KM,sulfite) of 512 µM ([Fe(tacn)2]3+)

and 970 µM ([Fe(dtne)]3+) were obtained (Supporting Information, Figure S6). Of course KM,sulfite should

be mediator-independent so these are not true Michaelis constants and they have little mechanistic

relevance other than defining the approximate linear current response of the electrode. The true KM,sulfite

value for HSO in solution is 9 µM in reaction with its physiological electron acceptor cytochrome c [41].

So utilizing the mass transport limitations presented by the membrane, the linear response of the

electrode to sulfite is increased by at least 2 orders of magnitude. We have observed similar

observations in other Mo enzyme systems [25, 26, 42].

3.5 HSO-Mediator Reaction

The HSO-mediator reaction was examined with increasing [Fe(tacn)2]3+ and [Fe(dtne)]3+

concentrations in the presence of a high (constant) concentration of sulfite. Figure 4A displays the effect

of varying the concentration of [Fe(tacn)2]3+ in the presence of 4 mM sulfite at a sweep rate of 5 mV s-1.

At 2 M [Fe(tacn)2]3+, a sigmoidal voltammogram is found, which is indicative of an electrochemical

steady state; the forward and backward sweeps are the same and the catalytic current is switched on

and off in a Nernstian fashion. However, as the concentration of [Fe(tacn)2]3+ increases (to 4 and 6 µM),

the waveform becomes asymmetric due to an excess of the oxidized form of mediator being produced

at the electrode, which overwhelms the limiting amount of HSOred formed during the sulfite oxidation

step. In other words the concentration of [Fe(tacn)2]3+ is no longer at steady state. Figure 4B displays

similar experiments but this time with increasing concentrations of [Fe(dtne)]3+ (6, 12 and 18 M). The

observed sigmoidal wave at 6 µM turns to a transient form upon increasing concentration to 12 and 18

15

µM [Fe(dtne)]3+. Data collected at all mediator concentrations are shown in Supporting Information

Figures S4B and S5B.

3.6 pH Dependence

The pH dependence of the catalytic sulfite oxidation current at the GC/HSO electrode was

explored in the range 5.5 < pH < 11 in 100 mM mixed buffer solution. Figure 5 depicts the baseline

subtracted maximum catalytic current as a function of pH. The actual CVs are provided in the Supporting

Information (Figure S7). The catalytic current exhibits a pH optimum of 8.5 which is similar to that

reported for HSO at an osmium redox polymer modified electrode [43] as well as in a solution assay for

HSO with its natural electron acceptor cytochrome c [44]. A bell shaped profile obtained by the

application of equation 3 enabled the two pKa values to be determined (7.2 and 9.8); the lower value

defining the protonation constant of a base that switches off catalysis and the higher one being the

protonation constant of a base that switches on catalysis. It has been proposed that Tyr343 plays an

important role in HSO catalysis involving substrate binding [45]. The higher pKa value observed here may

be due to Tyr343 deprotonation at high pH which is believed to be close to the active site and involved

in H-bonding with the substrate [45]. The pH profile was independent of the direction of titration and

catalytic activity was fully restored when the solution pH was returned to its optimal value. Furthermore

the voltammetry of both [Fe(tacn)2]3+ and [Fe(dtne)]3+ are pH-independent within this range. The

complex [Fe(tacn)2]3+ can be deprotonated but only at much higher pH (pKa 11.7) [46].

16

3.7 Electrochemical Simulation

In recent years, we have employed digital simulation for a better understanding of the

mechanism of mediated enzyme electrochemical reactions [26, 47-50]. The objective of the simulation is

to obtain the rate constants defined in Scheme 2 that reproduce all voltammetric features over a range

of sweep rates, substrate and mediator concentrations.

The voltammetric sweep rate is a significant variable to elucidate the kinetics of electrochemical

processes coupled with chemical reactions. The DigiSim program enables the same set of kinetic

parameters to be optimized to CVs measured across a range of sweep rates, but under an identical set

of concentrations (HSO, mediator and sulfite). When the concentrations of mediators and sulfite are

varied then ideally the same parameters reproduce CVs measured under those situations as well. Figure

6 shows experimental and simulated CVs for 800 µM sulfite in the presence of 10 µM [Fe(tacn)2]3+

(Figure 6A) and 20 µM [Fe(dtne)]3+ (Figure 6B) as a function of sweep rate. All other sweep rate

dependent simulated voltammograms recorded as a function of various mediator and substrate

concentrations are given in the Supporting Information (Figure S8-S13). The same features are well

reproduced for both mediators. In Figure 6A as the scan rate increases from 5 to 50 mV s-1, the

asymmetric transient CV becomes reversible as electrochemical oxidation and reduction of the mediator

is too rapid and the HSO-mediator reaction becomes uncompetitive. A very similar trend observed for

[Fe(dtne)]3+ as shown in Figure 6B and these features are also well reproduced in the simulation.

The substrate binding rate constant (k1) is well defined by simulation and changing its value has

a major influence on the quality of the fit between experiment and theory. Although k2 is also an

important value, and defines the maximum current at HSO saturation, its value is entangled with the

concentration of HSO under the membrane (equation 2, imax = nFAk2[HSO]). The concentration of HSO

under the membrane is only known approximately because the volume under the membrane cannot be

17

measured directly but instead determined by introduction of an known amount of external standard e.g.

cytochrome c as reported previously [25]. In this case k2 is the same as determined for HSO at pH 8.0

[45]. The product dissociation rate k3 value has little influence on the CV in this case if allowed to deviate

from its optimal value (values in the range 0.5 to 50 s-1 gave the same result here. The k4 values are also

accurately determined although the same issues regarding the accurate concentration of HSO under the

membrane introduce some uncertainty.

The CVs as a function of increasing mediator concentration of [Fe(tacn)2]3+ (1 to 4 µM) and

[Fe(dtne)]3+ (2 to 8 µM) in the presence of a saturating (4 mM) sulfite concentration and sweep rate of 5

mV s-1 are represented in Figure 7. An approximately sigmoidal wave is observed at a low concentration

of [Fe(tacn)2]3+ (1 µM) and this wave becomes progressively peak-shaped (transient) as the higher

concentration of mediator overwhelms the HSO present and the electrochemical steady state of

mediator breaks down.

The same set of parameters also reproduced CVs measured at various sulfite concentrations.

Figure 8 displays the anodic current response of the GC/HSO electrode as function of sulfite

concentration in the presence of 10 µM of [Fe(tacn)2]3+ (Figure 8A) and 20 µM of [Fe(dtne)]3+(Figure 8B).

At lower concentrations of sulfite (400 µM), the voltammograms took on a reversible transient form due

to the excess amount of oxidized mediator at the electrode surface. The wave becomes firstly

asymmetric as the sulfite concentration rises to 1600 µM, where catalysis becomes significant, and

tailing is due to sulfite depletion. Finally as the enzyme is saturated with sulfite (> 3 mM) the expected

steady state sigmoidal waveform is observed.

18

The catalytic current increased linearly with sulfite concentrations up to 800 and 1600 µM in the

presence of [Fe(tacn)2]3+ and [Fe(dtne)]3+, respectively. This discrepancy is due to the different driving

forces of the mediators (different k4 values) and the catalytic current attains saturation quickly when the

oxidative driving force is small ([Fe(tacn)2]3+) but extends to a much higher value when the driving force

is relatively high ([Fe(dtne)]3+). We have reported similar phenomena before in the mediated

electrochemistry of the enzyme DMSO reductase [49]. Briefly the more rapid reaction between HSO and

[Fe(dtne)2]3+ leads to faster sulfite depletion under the membrane and thus higher concentrations of

sulfite are needed to reach the point where the enzyme becomes truly saturated. An additional point of

interest is the low redox potential of [Fe(tacn)2]3+/2+ in these experiments (+56 mV vs NHE). This is similar

to the heme b redox potential reported for HSO [6, 41] but much lower than the natural redox partner

cytochrome c (ca. +260 mV). Thus the [Fe(tacn)2]3+:HSO catalytic system operates as an extremely low

catalytic potential (~200 mV lower than the naturally mediated electrochemical reaction), which is

advantageous for avoiding interference from the nonspecific oxidation of other species in solution.

3.8 Analysis of Kinetics Parameters

The rate and equilibrium constants defined in Scheme 2 and presented in Table 1 reproduced all

our experimental voltammetry carried out at different sweep rates, concentrations of sulfite and both

mediators. Importantly there same set of mediator-independent parameters (k1-k3) were used for the

two mediators. The accurate determination of multiple parameters is problematic in that some

parameters will have no effect on the CV depending on the concentrations of the reactants. That is the

rate limiting step in Scheme 2 will vary depending on the conditions. The sulfite binding rate constant

determined here (k1 = 106 M-1s-1) has not been reported for HSO. This value may even be an

underestimate due to mass transport limitations set by the membrane. The turnover number (k2 = 25 s-1)

19

obtained in the simulations is consistent with experimental value reported by solution assays for wild

type HSO with its natural electron acceptor cytochrome c in pH 8 (27 s-1) [41]. The larger outer sphere

electron transfer rate constant for the HSO:[Fe(dtne)]3+ reaction compared with the HSO:[Fe(tacn)2]3+

reaction is consistent with Marcus theory (log ket –G2) [51] given that higher redox potential of

[Fe(dtne)]3+ delivers a greater driving force. The rate constant (k4) obtained for the reaction between

the SO and higher potential mediator [Fe(dtne)]3+ is similar to the value reported for HSO in reaction

with cytochrome c (4.0 × 106 M-1 s-1) [23].

3.9 Amperometric Sulfite Determination

Sulfite is used as a preservative in food and beverages to prevent oxidation and bacterial growth

and to control enzymatic reactions during production and storage [27, 28]. Nevertheless, sulfite has

been regulated since the realization that it may cause asthmatic attacks and allergic reactions in some

people [52, 53]. Typically, a warning label is required for any food or beverage containing more than 10

ppm (125 M) sulfite so its accurate measurement in solution is important. Here we were able to

achieve sulfite determination using the GC/HSO electrode and the low potential mediator [Fe(tacn)2]3+ in

an amperometric experiment. Figure 9A illustrates the amperometric i-t curve for the catalytic

oxidation of sulfite at a GC/HSO electrode (covered with a dialysis membrane) in a homogeneously

stirred 50 mM Tris buffer solution (pH 8) at an applied potential of +150 mV vs NHE. An initial baseline

current response was stabilized for about 2 min in the presence of 10 µM [Fe(tacn)2]3+ at the GC/HSO

electrode to ensure the mediator was able to concentrate under the membrane (vide supra). Upon

addition of 10 µM of sulfite to the stirred solution in the electrochemical cell, the catalytic anodic

current increased suddenly and reached a plateau (steady state) within 3 sec. Further 10 µM sulfite

increments at intervals of 100 s led to a regular and consistent step in the current. Furthermore, the

20

amperometric current increased linearly with sulfite concentration from 10 to 180 µM and the detection

limit [54] was found to be 0.2 pM (S/N = 3) (Figure 9B). The obtained detection limit is even lower than

we reported with Starkeya novella sulfite dehydrogenase on a 11-mercaptoundecanol monolayer

modified Au electrode (44 pM) [25].

In separate voltammetry experiments, we found that the anodic catalytic current increased

linearly from 5 to 800 µM at +150 mV vs NHE (Supporting Information, Figure S6). Spricigo et al.

reported a sulfite biosensor by co-immobilization of HSO within an osmium redox polymer on a carbon

screen-printed electrode [43]. The biosensor operates at +100 mV vs NHE with detection limit (0.5 µM)

and linearity (1 to 100 µM).

3.10 Determination of Sulfite in Wine and Beer Samples

In order to demonstrate the practical application of the present biosensor, the enzyme modified

electrode was used for the determination sulfite concentration in white wine and beer samples. Two

beers and one white wine sample were obtained commercially were analyzed for sulfite using the

present biosensor and this was validated by the standard spectroscopic method [37] using Ellman’s

reagent.

Beer and wine, as prepared, are acidic (~pH 4) and at this pH HSO is inactive as shown in Figure

5. Therefore, the beer and wine samples were neutralized with dilute NaOH then diluted with Tris buffer

solution and analyzed immediately without any other pretreatment. The method of standard additions

was employed by injecting known amounts of sulfite to each beer or wine sample within the linear

range and measuring the increase in catalytic current which enabled the original sulfite concentration to

be determined by back extrapolation to zero current (Supporting information, Figure S14) . Further, we

21

did not observe any interference signals in the beer and wine samples from non-specific oxidation

reactions at the electrode. Table 2 shows the results of sulfite determination in beer and wine sample

using the present electrochemical biosensor. The obtained results are compared with the standard

spectroscopic method (Supporting information, Figure S15) where sulfite reacts with Ellman’s reagent

(5,5’-dithio-bis(2-nitrobenzoic acid) to produce an organic thiosulfate and releasing coloured 5-

mercapto-2-nitrobenzoate. The sulfite concentration determined using the present electrochemical

biosensor is in excellent agreement with the spectroscopic method. The obtained results clearly

revealed that the present electrochemical biosensor is suitable for practical applications.

4. Conclusions

We have demonstrated the mediated catalytic voltammetry of HSO with two synthetic electron

acceptors [Fe(tacn)2]3+ and [Fe(dtne)]3+. The redox potential difference between these two mediators

results in different oxidative driving forces for enzyme catalysis. A set of self-consistent rate constants

was obtained by simulating the experimental CVs measured at different sweep rates, mediator

concentrations and substrate concentrations. An amperometric biosensor was constructed with the

lower potential mediator [Fe(tacn)2]3+and it showed linear catalytic response from 5 µM to 800 µM

sulfite and lowest detection limit of 0.2 pM (S/N = 3). As a practical application of the HSO modified

electrode, we successfully used it for the amperometric determination of sulfite concentration in beers

and wine samples and the results agreed well with values obtained by a standard spectroscopic method.

Acknowledgements

PVB acknowledges financial support from the Australian Research Council (DP150103345).

22

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24

Figure 1. Molecular structures and redox potentials of the mediators used in this study.

25

–200 0 200

0

1

/ mV vs. NHEE

Ia

b

Figure 2. CVs obtained for 10 µM [Fe(tacn)2]3+ in the absence (red) and presence (green) of 5 mM sulfite

at the GC/HSO electrode in 50 mM Tris buffer (pH 8) at a sweep rate of 5 mV s-1.

26

Figure 3. CVs obtained for varying sulfite concentrations in the presence of (A) 10 µM [Fe(tacn)2]3+ and (B) 20 µM [Fe(dtne)]3+ at GC/HSO electrode in 50 mM Tris buffer (pH 8) at a sweep rate of 5 mV s-1.

–200 0 200

/ mV vs. NHEE

A

200 400 600

/ mV vs. NHEE

B

27

Figure 4. CVs obtained for varying (A) [Fe(tacn)2]3+ and (B) [Fe(dtne)]3+ concentrations in the presence of 4 mM sulfite at GC/HSO electrode in 50 mM Tris buffer solution (pH 8) at a sweep rate of 5 mV s-1.

–200 0 200

/ mV vs. NHEE

A

200 400

/ mV vs. NHEE

B

28

5 6 7 8 9 10 11

0.2

0.4

0.6

0.8

1.0

1.2

I /

A

pH

Figure 5. Plot of the pH dependence of the maximum catalytic oxidation current at the GC/HSO electrode with 4 mM sulfite and in the presence of 10 µM [Fe(tacn)]2

3+ in 100 mM mixed buffer solution at a scan rate of 5 mV s-1. The solid curve is obtained from a fit to the experimental points using equation 3 (pKa1 9.8 and pKa2 7.2).

29

–200 0 200

/ mV vs. NHEE

5 mV s–1

10 mV s–1

20 mV s–1

50 mV s–1

A

200 400

/ mV vs. NHEE

5 mV s–1

10 mV s–1

20 mV s–1

50 mV s–1

B

Figure 6. Experimental (solid lines) and simulated (broken lines) sweep rate dependent CVs obtained for 800 µM sulfite in the presence of (A) 10 µM [Fe(tacn)2]3+ and (B) 20 µM [Fe(dtne)]3+ at GC/HSO electrode in 50 mM Tris buffer solution (pH 8) at different scan rates.

30

–200 0 200

/ mV vs. NHEE

A

200 400

/ mV vs. NHEE

B

Figure 7. Experimental (solid lines) and simulated (broken lines) CVs obtained for varying mediator concentration in the presence of 4 mM of sulfite (A) [Fe(tacn)2]3+ and (B) [Fe(dtne)]3+ at the GC/HSO electrode in 50 mM Tris buffer solution (pH 8) at a sweep rate of 5 mV s-1.

31

200 400

/ mV vs. NHEE

B

–200 0 200

/ mV vs. NHEE

A

Figure 8. Experimental (solid lines) and simulated (broken lines) CVs obtained for varying sulfite

concentration in the presence of (A) 10 µM of [Fe(tacn)2]3+ and (B) 20 µM of [Fe(dtne)]3+ at the GC/HSO

electrode in 50 mM Tris buffer solution (pH 8) at a sweep rate of 5 mV s-1.

32

1000 20000

100

200

Time (sec)

I /n

A

A

0 20 40 60 80 100 120 140 160 180

0

20

40

60

80

100

120

140

160

180

200

y = 1.202x - 2.015

R2 = 0.9998

B

I /

nA

Concentration (M)

Figure 9. (A) An amperometric i−t curve obtained for the determination of sulfite at the GC/HSO electrode in stirred 50 mM Tris buffer solution (pH 8). Each increment corresponded to a 10 µM increase in sulfite which was injected at regular intervals of 100 s. The electrochemical cell contained 10 µM [Fe(tacn)2]3+ and the electrode was poised at +150 mV vs NHE. (B) Plot of the steady state current as a function of sulfite in the linear range.

33

Scheme 1. Simplified catalytic cycle of SO reduced forms of enzyme and substrate in red and oridised

forms of enzyme and product in blue.

34

Scheme 2. Mediated Electrochemically Driven Catalysis of HSO.

35

Table 1. Kinetic parameters (defined in Scheme 2) from electrochemical simulation.

a k4 = k4’; b k-4 = k-4’; c approximate (simulation not sensitive to this parameter); d KM,sulfite = (k2 + k−1)/k1.

E′ (mV vs NHE) [Fe(tacn)2]3+

56 mV [Fe(dtne)]3+

355 mV

k4 (M-1 s-1)a 1.0 × 104 2.0 × 106 Mediator dependent k-4 (M-1 s-1)b 0.1c 2c

k1 (M-1 s-1) 1.0 × 106

Mediator independent

k-1 (s-1) 20 k2 (s-1) 25 k-2 (s-1) 5.0 × 10-2 c k3 (s-1) 5c

k-3 (M-1 s-1) 1.0 × 10-2 c

KM,Sulfite (µM)d 102

36

Table 2. Determination of sulfite in wine and beer samples using present method and also compared with the standard spectroscopic method

Sample Present methoda (µM)

RSD (%)

Spectroscopic methoda (µM)

RSD (%)

Beer sample 1 600 2.2 588 2.0

Beer sample 2 540 2.0 532 2.2

White wine sample 1250 1.8 1240 2.0

a Mean of three determinations