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Accepted Manuscript Title: Low Potential Catalytic Voltammetry of Human Sulte Oxidase Author: Palraj Kalimuthu Abdel A. Belaidi Guenter Schwarz Paul V. Bernhardt PII: S0013-4686(16)30180-3 DOI: http://dx.doi.org/doi:10.1016/j.electacta.2016.01.181 Reference: EA 26563 To appear in: Electrochimica Acta Received date: 10-12-2015 Revised date: 21-1-2016 Accepted 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 Sulte Oxidase, Electrochimica Acta http://dx.doi.org/10.1016/j.electacta.2016.01.181 This is a PDF le of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its nal form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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  • 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

    This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.


  • 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: [email protected]

  • 2

    –200 0 200



    / mV vs. NHEE




    Graphical Abstract


    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


    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


    𝐾M,app + [SO32−]


    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


    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


    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.


    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



    / mV vs. NHEE



    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


    200 400 600

    / mV vs. NHEE


  • 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


    200 400

    / mV vs. NHEE


  • 28

    5 6 7 8 9 10 11







    I /



    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)]23+ 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


    200 400

    / mV vs. NHEE

    5 mV s–1

    10 mV s–1

    20 mV s–1

    50 mV s–1


    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


    200 400

    / mV vs. NHEE


    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


    –200 0 200

    / mV vs. NHEE


    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



    Time (sec)

    I /n



    0 20 40 60 80 100 120 140 160 180












    y = 1.202x - 2.015

    R2 = 0.9998


    I /


    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