Catalytic Voltammetry of the Molybdoenzyme Sulfite Dehydrogenase

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Catalytic Voltammetry of the Molybdoenzyme Sulfite Dehydrogenase from Sinorhizobium meliloti Palraj Kalimuthu, Ulrike Kappler, and Paul Vincent Bernhardt J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/jp503963z • Publication Date (Web): 03 Jun 2014 Downloaded from http://pubs.acs.org on June 15, 2014

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The Journal of Physical Chemistry

Catalytic Voltammetry of the Molybdoenzyme Sulfite Dehydrogenase from Sinorhizobium meliloti

Palraj Kalimuthu, Ulrike Kappler and Paul V. Bernhardt*

School of Chemistry and Molecular Biosciences,

University of Queensland, Brisbane, 4072, Australia

*Corresponding author: Fax: + 61 7 3365 4299; E-mail: [email protected] 1

ACS Paragon Plus Environment


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Abstract Sulfite dehydrogenase from the soil bacterium Sinorhizobium meliloti (SorT) is a periplasmic, homodimeric molybdoenzyme with a molecular mass of 78 kDa. It differs from most other well studied sulfite oxidizing enzymes as it bears no heme cofactor. SorT does not readily reduce ferrous horse heart cytochrome c which is the preferred electron acceptor for vertebrate sulfite oxidases. In the present study, ferrocene methanol (FM) (in its oxidized ferrocenium form) was utilised as an artificial electron acceptor for the catalytic SorT sulfite oxidation reaction. Cyclic voltammetry of FM was used to generate the active form of the mediator at the electrode surface. The FM-mediated catalytic sulfite oxidation by SorT was investigated by two different voltammetric methods namely (i) SorT freely diffusing in solution and (ii) SorT confined to a thin layer at the electrode surface by a semi-permeable dialysis membrane. A single set of rate and equilibrium constants was used to simulate the catalytic voltammograms performed under different sweep rates and with various concentrations of sulfite and FM which provides new insights into the kinetics of SorT catalytic mechanism. Further, we were able to model the role of the dialysis membrane in the kinetics of the overall catalytic system. Keywords: enzyme; electrochemistry; molybdenum; mediator; simulation

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Introduction The electrochemistry of redox enzymes is now a mature area of research spurred on by both fundamental advances in our understanding of long range electron transfer and practical applications such as biosensor development.1-5 Unlike small molecule electrochemistry, the redox active cofactors (including the active site) are often deeply buried below the surface of the protein and this can prohibit direct (heterogeneous) electron transfer with the electrode. Further, direct electron transfer may be problematic due to ensuing denaturation of the enzyme which is typically physisorbed on the working electrode. Alternatively, electron transfer can be assisted by a redox mediator either in solution6, 7 or immobilized on an electroactive layer at the electrode surface.8, 9 Understanding these reactions can provide fundamental insight into physiological electron transfer processes as well as providing impetus for the development of novel amperometric biosensors and bioelectrocatalytic systems. Specific to this work is the enzyme sulfite dehydrogenase. The molybdenum dependent sulfite oxidising enzymes are common to bacteria, plants and animals and detoxify highly reactive sulfite, which is produced either as part of cellular metabolism or by environmental processes, by oxidation to chemically inert sulfate.10 To illustrate the importance of these enzymes, a deficiency in the human sulfite oxidase (leading to a build-up of sulfite) results in severe neonatal neurological problems and early death if untreated.11, 12 Structurally the sulfite oxidising enzymes (regardless of origin) share a common active site comprising a dioxido-MoVI moiety chelated by a molybdopterin dithiolene ligand in addition to a cysteinyl S-donor (Scheme 1).13 The equatorial oxido ligand is the one transferred to sulfite during its 2electron O-atom transfer reaction. The diversity of structure amongst the sulfite oxidising enzymes emerges principally from the presence (or absence) of an additional heme cofactor as well as the existence of monomeric and hetero- or homo-dimeric structures. The three most intensively 3



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investigated sulfite oxidising enzymes (chicken sulfite oxidase, human sulfite oxidase and the bacterial sulfite dehydrogenase from Starkeya novella) all bear a heme cofactor which accepts electrons (one at a time) from the Mo active site after sulfite oxidation and it is this site that is in turn oxidized by the native electron partner. Sulfite oxidase from the plant Arabidopsis thaliana contains no redox active center other than the Mo active site and in this enzyme the reduced MoIV cofactor donates electrons directly to dioxygen after turnover, leading to the production of hydrogen peroxide as the final product.14-16 Although the lack of a heme cofactor in this plant sulfite oxidase seemed unusual at the time, more recent studies on bacterial sulfite dehydrogenases have revealed other examples. The sulfite dehydrogenase from the Gram-negative soil bacterium Sinorhizobium meliloti (the enzyme hereafter referred to as SorT), has recently been characterized and interestingly does not have sulfite oxidase activity (the ability to reduce molecular oxygen).17, 18 A potential natural electron acceptor, the c-type cytochrome, SorU (Smc04048), for SorT has been identified.18 O3PO

O3PO O HN N H2N

S Mo

NH O

NH

O S

VI

O S-Cys O

-e , -H

H2N

SO32-

SorTox

-

HN N NH

O3PO O HN N H2N

O

MoIV

S

SorTred

NH

NH O

O S

MoV

S

S-Cys OH

SO42-

SorTint

Scheme 1. Catalytic Oxidation of Sulfite at the Active Site of SorT.

4


S-Cys OSO3

-e-, -H+

+

SO32-

NH

O S

H2O

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The general mechanism for enzymatic sulfite oxidation by SorT (and all other known sulfite oxidoreductases) is shown in Scheme 1.13 Nucleophilic attack by sulfite on the equatorial oxido ligand (SorTox in its MoVI form) is coupled with 2-electron reduction to MoIV.19, 20 The sulfato ligand is liberated from SorTred by hydrolysis and electron transfer to an external electron acceptor then restores the enzyme to the active MoVI form, usually in two single electron steps via the MoV intermediate SorTint. Proton transfer reactions accompany these redox processes as shown. SorT is a homodimer consisting of a Mo-binding domain and a (redox inactive) dimerization domain.17, 18 The activity of the recombinant purified SorT was tested with the electron acceptors horse heart cytochrome c (Eo’ +260 mV vs NHE) and ferricyanide (Eo +380 mV vs NHE) and found to be 7.4 U/mg and ~470 U/mg, respectively at pH 8.18 The redox potential of the SorU (Smc04048) cytochrome, the natural electron acceptor for SorT was reported as +205 mV (pH 6.9 from cyclic voltammetry),18 and it emerges that SorU is essentially a substitute for the heme subunit (SorB) in the well-studied bacterial heterodimeric sulfite oxidising enzyme (SorAB) from S. novella.21,

22

Favourable protein-protein

interactions between SorT and SorU lead to productive electron transfer and catalytic activity (33.4 U/mg)18 while ferric horse heart cytochrome c, although a more powerful oxidant, is unable to bind effectively to SorT and activity is lowered (7.4 U/mg) as a consequence. The above success of ferricyanide as an artificial electron partner for SorT illustrates that small molecular weight compounds may overcome molecular recognition problems inherent to protein:protein electron transfer. This prompted us to turn to small molecules of similar redox potential to act as mediators of electron transfer between the electrode and SorT. Herein, we report the mediated

electrocatalytic

voltammetry

of

SorT

with

the

high

redox

potential

complex

ferrocenium/ferrocene methanol (FMox/red, +430 mV vs NHE, Scheme 2) at a glassy carbon working electrode. Digital simulation was employed to explore the kinetics of the coupled homogeneous chemical reactions at a variety of sweep rates as well as substrate and mediator concentrations, a 5



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method we have used to advantage in recent studies on the Mo enzymes DMSO reductase,7, 23 xanthine dehydrogense,6, 24 arsenite oxidase25 and nitrate reductase.26

Scheme 2. Mechanism of electrochemically mediated, SorT catalysed sulfite oxidation.

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Experimental Section Materials. Sulfite dehydrogenase from Sinorhizobium meliloti was purified following heterologous expression in E. coli as previously described.18 Ferrocene methanol (FM)27 was synthesized by NaBH4 reduction of commercially available ferrocene carbaldehyde in MeOH washing with water then extraction of the product into chloroform. The product was of satisfactory purity by 1H NMR. All other reagents used were of analytical grade purity and were used as supplied. All solutions were prepared in purified water (Millipore, resistivity 18.2 MΩ.cm). Tris (acetate) buffer (50 mM) was used for experiments at pH 8. For experiments performed across a range of pH a mixture of buffers was used comprising 10 mM MES, 10 mM Bis-Tris, 10 mM Tris, 10 mM CHES and 10 mM CAPS while the desired pH was obtained by addition of dilute acetic acid or NaOH. MEMBRA-CEL® dialysis tubing (regenerated cellulose, MWCO 3500 Da) was used as supplied, cut into small squares (ca. 3 cm2) and soaked briefly in distilled water before use. The membranes are stable for extended periods within the range 5 < pH < 9 as long as they are kept moist.

Electrochemical Measurements and Electrode Cleaning. Cyclic voltammetry (CV) was carried out with a BAS 100B/W electrochemical workstation using a three-electrode system consisting of a glassy carbon (GC) working electrode, a platinum wire counter electrode, and a Ag/AgCl reference electrode (+196 mV vs NHE). Potentials are cited versus NHE. Unless otherwise stated, electrochemical solutions were purged with argon for at least 30 min prior to measurement and all experiments were performed under a blanket of argon thereafter. The GC electrode was polished first with 0.50 then 0.05 μm alumina slurry and rinsed thoroughly with water after each polish. The electrode was then sonicated in water for 5 min to remove adsorbed alumina particles and dried in a stream of argon. No other electrode surface conditioning was necessary.

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The electro-active surface area of the GC electrode (A, cm2) was determined by CV analysis of 1 mM FM in aqueous 0.1 M KCl supporting electrolyte at different sweep rates using the Randles-Sevcik equation (eq. 1).28 The published diffusion coefficient (Do) of ferrocene methanol is 6.7 × 10-6 cm2 s-1,29 ip is the measured current maximum (A), n is the number of electrons, Co is the FM concentration (mol cm-3) and ν is the sweep rate (V s-1). ip = (2.69 × 105)n3/2ADo1/2Coν1/2

(1)

The variation of the observed limiting catalytic current (ilim) as a function of sulfite concentration followed Michaelis-Menten kinetics and the data were fit to equation 2.30 =

[SO ] (2) , + [SO ]

where imax is the catalytic current at saturation and KM,app is the apparent Michaelis constant.

Enzyme Electrode Preparation. Catalytic voltammetry of SorT was carried out by two different methods (i) SorT, FM and sulfite all in solution (ca. 500 μL) and under diffusion control and (ii) SorT confined to a thin layer near the CG electrode surface by a dialysis membrane while FM and sulfite are in solution (10 mL) and may cross the membrane to react with SorT and the electrode as follows. A 5 µL aliquot of 117 µM SorT in Tris buffer (pH 8) was dispensed onto a freshly polished, inverted GC electrode and this was allowed to dry to a film at 4 oC. The electrode surface was carefully covered with a dialysis membrane (~3 cm2, MW cutoff 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 enzyme solution under the membrane. The resulting modified electrode was stored at 4 oC in 50 mM Tris buffer (pH 8.0) when not in use. This method has advantages such as requiring minimal amounts of enzyme and also the electrode may be removed from solution and reused many times.31

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Digital Simulation. DigiSim (version 3.03b) program was employed to simulate the experimental cyclic voltammograms.32 The experimental parameters restrained in each case were the working electrode surface area (0.055 cm2 from eq. (1)) and the double-layer capacitance (2 µF). Semiinfinite diffusion was assumed and all pre-equilibration reactions were disabled. The diffusion coefficients of FM (4 × 10-6 cm2 s-1)29 and sulfite (1 × 10-5 cm2 s-1)33 (both under diffusion control in solution) were comparable with values in the literature (small variations may be attributed to different supporting electrolytes and ionic strength used here) while that for SorT (in all of its forms) was estimated as 1×10-7 cm2 s-1; a value consistent the size of the protein and with our previous investigations of other enzymes from the molybdoenzyme family.6, 7, 23, 25, 26 In the presence of a dialysis membrane covering the electrode the FMox/FMred and sulfite/sulfate molecules must cross the membrane and this brings a mass transport limitation to their diffusion toward/away from the reaction layer. This was modelled by using diffusion coefficients that were approximately one order of magnitude smaller (see Table 1). The heterogeneous rate constant (k0) was 0.05 cm s-1 was determined from simulating the CV of FM as a function of sweep rate. The apparent redox potential of the FMox/red couple (+430 mV vs NHE) was determined in the absence of SorT and sulfite. All of the above-mentioned parameters were then held constant during CV simulations of experiments performed at different scan rates, sulfite concentrations and FM concentrations so only the homogenous rate constants in Scheme 2 (k1/k-1, k2/k-2, k3/k-3, k4/k-4 and k’4/k’-4) were optimized. These values appear in Table 1.

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Results and Discussion Experimental Voltammetry Mechanism. The simplified double substrate (ping-pong) mechanism is employed to model the reaction kinetics. The substrate (sulfite) and mediator (FM) are under diffusion control in solution. SorT reacts with both sulfite and FM but does not undergo any heterogeneous electron transfer reactions. We have assumed that the SorT-sulfite reaction follows Michaelis-Menten kinetics comprising reversible sulfite binding (k1/k-1), turnover (k2/k-2) and sulfate release (k3/k-3). The homogeneous SorT-FM reactions (k4/k-4 and k’4/k’-4 in Scheme 2) are treated as pure second order outer-sphere electron transfer processes with no pre-equilibrium i.e. no SorTred:FMox outer sphere encounter complex is formed prior to electron transfer. If such a situation was occurring then the observed rate of the SorTred-FMox reaction would saturate at high FM concentrations. Practically this is very difficult to observe as at high FM concentrations ([FM] » [SorT]), the catalytic sigmoidal wave becomes overwhelmed by the standard reversible transient response of the FMox/red couple. Finally, due to the high driving force, the electron transfer reaction is effectively irreversible (k-4 « k4 and k’-4 « k’4).

Catalytic Voltammetry. An example of the mediated catalytic voltammetry of the SorT/sulfite/FM system with all species freely diffusing (no dialysis membrane) is illustrated in Fig. 1. In the control experiment, with only a 50 µM solution of FM (pH 8) a reversible FMox/red redox wave is seen at +430 mV (vs NHE) with a peak to peak separation of 60 mV (Fig. 1a). Introduction of 200 µM sulfite to the electrochemical cell leads to a small increase in the anodic current at the high potential end of the sweeps (~+550 mV) which is associated with the onset of non-specific sulfite oxidation at the bare GC electrode (peaking at ~600 mV vs NHE, data not shown).34 Note that the FMox/red current is unaffected, proving that FMox does not oxidize sulfite, as expected. Upon introduction of the enzyme SorT (10 µM) into the electrochemical cell (in the presence of both 50 μM FM and 200 µM sulfite) a well-defined 11



sigmoidal wave emerges and the cathodic peak vanishes (curve c) which is indicative of a catalytic homogeneous reaction coupled to heterogeneous electron transfer (EC′ mechanism).28 Here sulfite is oxidized enzymatically yielding SorTred which is reoxidized by electrogenerated FMox. Note that the catalytic half-wave potential coincides with that of the FMox/red couple and is ca. 200 mV lower than the potential of non-specific sulfite oxidation. The sigmoidal shape of the CV in Fig. 1c is characteristic of an electrochemical steady state where FMox generated at the electrode surface is consumed by SorTred at the same rate i.e. the concentration of FMox at the electrode surface is constant at a given potential (but of course potential dependent according to the Nernst equation). c 0.6

0.4

I / µΑ

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

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b a

0.2

0

300

400

500

600

E / mV vs. NHE Fig. 1. CVs obtained for (a) 50 μM FM (b) 50 μM FM in the presence of 200 µM sulfite and (c) 10 µM SorT, 50 μM FM and 200 µM sulfite. GC electrode (no membrane), 50 mM Tris buffer (pH 8) and sweep rate 5 mV s−1.

SorT-Sulfite Reaction. The SorT-sulfite reaction was examined by varying the sulfite concentration in the presence of constant FM and SorT concentrations both in the absence (Fig. 2A) and presence (Fig. 2B) of a dialysis membrane covering the electrode and trapping SorT at the electrode surface. At 25 µM sulfite (with no membrane), an asymmetric catalytic transient wave emerges in the presence of 50 µM FM and 10 µM SorT (Fig. 2A, curves b, c and d). The ‘tailing’ of the anodic peak is due to depletion of sulfite (consumed by SorT) at a rate faster than it can diffuse to the enzyme active site so

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the electrochemical steady state breaks down as the system is uncoupled and the voltammetry reverts to that of FM alone. Note that the cathodic wave of FM gradually diminishes as the sulfite concentration increases because the redox reaction becomes biased exclusively toward FM (and sulfite) oxidation. As the sulfite concentration rises depletion is no longer an issue and while the catalytic current continues to grow, the waveform becomes sigmoidal and more symmetrical (Fig. 2A, curve e); characteristic of an electrochemical steady state. At this point the diffusion layer narrows to a point where FMox is consumed by SorTred at a rate that exactly compensates for its production at the electrode (Scheme 2). 1

A

Absence of dialysis membrane

B

0.5

I / µΑ

0.1

e

I / µΑ

Presence of dialysis membrane

e a

0

k

0.5

a

I / µΑ

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


300

400

500

600

E / mV vs. NHE

a

0

0 300

400

500

600

300

E / mV vs. NHE

400

500

600

E / mV vs. NHE

Fig. 2. (A) CVs obtained for (a) 0, (b) 25, (c) 50, (d) 100 and (e) 200 µM sulfite in the presence of 50 µM FM and 10 µM SorT at GC electrode (no membrane) (B) CVs obtained for (a) 0, (b) 10, (c) 25, (d) 50, (e) 100, (f) 200, (g) 400, (h) 800, (i) 1200, (j) 1600 and (k) 2000 µM sulfite in the presence of 50 µM FM at GC/SorT membrane electrode (5 µL of 117 µM SorT under the membrane). 50 mM Tris buffer (pH 8) and sweep rate 5 mV s-1.

We repeated this experiment but in this case with an aliquot of SorT (5 µL of 117 µM SorT) confined to a thin layer at the electrode surface underneath a dialysis membrane. As shown in previous studies,35 this leads to an approximate five-fold concentration of SorT under the membrane to 600 µM within the small volume under the membrane (from an original concentration of 117 µM). As shown in Fig. 2B (inset), at low sulfite concentrations (< 100 µM) a pre-wave is observed at ~+400 mV in addition to the transient CV response of FM at +430 mV. The pre-wave grows steadily in magnitude with 13



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increasing sulfite concentration and gradually replaces the higher potential (transient) FM redox wave which completely disappears at 200 µM sulfite. At low sulfite concentration the pre-wave is associated with the coupled catalytic SorT-sulfite reaction but rapid depletion of sulfite in the vicinity of the electrode halts catalysis and the CV reverts to that of the uncoupled FMox/red response at ca. +430 mV when the supply of sulfite is exhausted. The principal reason that the pre-wave is more obvious in Fig. 2B than Fig. 2A is that the rate of sulfite mass transport to SorT (under the membrane) is slowed by its passage across the membrane and this effectively desensitizes the system to sulfite i.e. the dynamic response increases and the changes become more gradual. The theory behind this phenomenon has been discussed elsewhere.36 Similar findings were reported by our group recently with the cytochrome c552 mediated voltammetry of NT-26 arsenite oxidase where mass transport limitations on substrate (arsenite) diffusion to the enzyme attenuate the catalytic current.25 In this case, with the membrane present, the catalytic wave does not saturate until concentrations of ~2 mM sulfite are reached (Fig. 2B) compared with 200 µM in the absence of the membrane (Fig. 2A). The anodic current measured at +440 mV vs NHE without a membrane present increases linearly with sulfite concentrations only until 50 μM (correlation coefficient 0.9994) while with a membrane present the current at +440 mV increases linearly up to 800 μM sulfite (correlation coefficient 0.9924) (see supporting information Fig. S1). The ‘brute force’ application of eq. 2 to the raw voltametric data (limiting current as a function of sulfite concentration), gives widely different KM values (27 µM with no membrane and 847 µM with a membrane) due to the extended linear response range that is a consequence of the mass transport limitations of the membrane (Supporting Information Figs S1A and S1B). Strictly eq. 2 does not apply to

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mediated enzyme voltammetry experiments as the current response from the mediator is a combination of both diffusional and steady state (catalytic) components and the actual equation involves the outer sphere (FM-SorT) electron transfer rate constant as well.1 The key point is that the extended dynamic range in the presence of a membrane is a potentially useful feature in terms of application of this enzyme as a sulfite biosensor. The very high KM,app value (847 µM) in the presence of a membrane is discussed in greater detail in later sections. We recently reported similar observations for the catalytic voltammetry of NT-26 arsenite oxidase,25 nitrate reductase26 and xanthine dehydrogenase,24,

37

with very high KM,app values being observed in the presence of a membrane

covering the reactive enzyme layer.

0.4

A

B



0.4

0.2

f

I / µΑ

f

I / µΑ

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


0.2

a

a

0

0 300

400

500

600

300

E / mV vs. NHE

400

500

600

E / mV vs. NHE

Fig. 3. CVs obtained for varying FM concentrations of (a) 0, (b) 5, (c) 10, (d) 15, (e) 20 and (f) 25 μM (A) in the presence of 500 µM sulfite and 10 µM SorT at a GC electrode (B) in the presence of 500 µM sulfite at the GC/SorT membrane electrode (5 µL of 117 µM SorT). 50 mM Tris buffer pH 8 and sweep rate 5 mV s−1.

SorT-FM Reaction. The reaction between SorT and FMox was investigated by varying the solution FM concentration in the absence and presence of a dialysis membrane. Fig. 3A (no membrane) illustrates the effect of an increasing FM concentration in the presence of 10 µM SorT and 500 µM 15



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sulfite. In the absence of FM, no significant faradaic response was seen in the potential window +250 to +600 mV (Fig. 3A, curve a). This shows that no direct electrochemistry of SorT occurs at the GC electrode. However, a sigmoidal catalytic wave emerges on addition of 5 µM FM (curve b, with 500 µM sulfite and 10 µM SorT present) this wave increases steadily in amplitude with FM concentration (10-25 µM). It is apparent that very similar responses are seen in the presence of dialysis membrane (Fig. 3B). However, at higher concentration of FM (25 µM, curve f) the sigmoidal wave is slightly asymmetric (the anodic and cathodic sweeps do not overlay) which is due to slowed mass transport of FM across the membrane. pH Profile. The pH dependence of the catalytic current from the GC/SorT enzyme electrode (5 µL of 117 µM SorT under the membrane) was investigated in the presence of 800 µM sulfite and 50 µM FM at a sweep rate of 5 mV s-1. The voltammetry of FM alone exhibits no pH dependence in the range 4 < pH < 10 (data not shown) and non-specific sulfite oxidation is insignificant (Supporting Information Fig. S2) so any variations in catalytic current are attributable to protonation reactions affecting the SorTsulfite reaction. The results are shown in Fig. 4A and 4B. The catalytic current/pH profile is highly unusual and decreases almost linearly from pH 10 (maximum) to pH 5.5. It was found that in spite of high activity at pH 10, significant degradation of SorT occurs while the enzyme is poised at this high pH. This was established by the periodic measurement of catalytic activity at pH 8 after each pH step (Supporting Information Fig. S3). As shown in Fig. S3 the greatest loss in activity occurs while the enzyme is at pH 10 while in the range 6 < pH < 9 there is no significant variation. As seen in Fig. 4B the pH dependent activation/deactivation is totally reversible when partial degradation of the enzyme is taken into account by scaling the currents relative to the pH 8 ‘standards’ measured periodically (Fig. S3). Separate experiments performed at pH 11 and 12 showed even greater enzyme degradation and

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correction for this marked loss of activity was not practical so all experiments were performed at pH 10 or below.

A 0.4 0.3

0.6 pH 9 pH 8 pH 7 pH 6 pH 5.5

B from pH 5.5 to pH 10 from pH 10 to pH 5.5

0.5 0.4 Ilim / µA

0.5

I / µA

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


0.2

0.3 0.2

0.1

0.1

0.0

0.0

-0.1 300

400

500

5

600

6

E / mV vs. NHE

7

8 pH

9

10

11

Fig. 4. (A) CVs obtained for 800 µM sulfite and 50 µM FM at the GC/SorT electrode (5 µL of 117 µM SorT under the membrane) at a scan rate of 5 mV s-1 at different pH values and (B) plot of baseline subtracted limiting current as a function of pH (circles show titration from pH 5.5 to pH 10 and squares show reverse titration back to pH 5.5).

The lack of a plateau in Fig. 4B is that would be indicative of a genuine pH optimum and an apparently linear decrease in activity as a function of pH is not interpretable by standard models of pHdependent enzyme activity which predict sigmoidal or bell-shaped profiles. Although still not understood, these results are in accord with the published pH dependence of the SorT sulfite oxidation reaction measured by solution assays.18 The irreversible time-dependent loss of catalytic activity at pH 11 was also seen in solution assays over course of about 20 min at room temperature. This is similar to the timescales of each experiment performed at a given pH.

Electrochemical Simulation 17



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In order to understand the kinetics of the catalytic cycle quantitatively we have determined the rate constants defined in Scheme 2 using electrochemical simulation to reproduce all voltammetry performed at different sweep rates, sulfite concentrations and FM concentrations. The sweep rate is a powerful and easily controlled variable as it defines the timescale of the experiment and the homogeneous reactions in Scheme 2 can be uncoupled from the heterogeneous electron transfer reaction at rapid sweep rates. Similarly as many of the reactions in Scheme 2 are first order in [FM] or [SO32-] variations in either of these concentrations will have a major effect on the rate of the overall catalytic cycle i.e. current. However, it is important to highlight the fact that CV experiments with and without a membrane cannot be modelled with the same set of parameters (k0, k1/k-1 … k4/k-4, diffusion coefficients etc.). As mentioned before, both sulfite and FM must cross the membrane so the rates of any bimolecular reactions involving these species will be attenuated. The parameters determined without a membrane are clearly the more accurate ones as no limitations on diffusion are present. However, it should be emphasized that the rate constants k1/k-1, k2/k-2, k3/k-3, k4/k-4 and k’4/k’-4 are intrinsic to each chemical reaction and should not be dependent on whether a membrane is present or not. Mass transport limitations due the presence of the semi-permeable membrane can be modelled by variations in the ‘apparent’ diffusion coefficients in the presence or absence of a membrane. The membrane solute permeability coefficient is directly proportional to the diffusion coefficient;38 both being proportional to the flux of the solute in this case toward the enzyme trapped under the semi-permeable membrane adjacent to the electrode. The membrane thickness and permeability are constant for each experiment and thus the use of smaller diffusion coefficients for FM and sulfite is a simple way of modelling the changes in mass transport rate due to passage across the membrane. All of the simulation parameters are collected in Table 1. By separating the bimolecular reaction rate constants (k1/-1 to k4/-4) from mass

18


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transport control we are able to derive a single set of rate constants that cover all of the experimental conditions (different concentrations of FM, sulfite and across a range of sweep rates).

A


0.4 µΑ

–1 2 mV s

B


–1 2 mV s

0.1 µΑ

–1 5 mV s

–1 5 mV s –1 10 mV s

–1 10 mV s

–1 20 mV s

–1 20 mV s

0.2 µΑ –1 50 mV s

300

–1 50 mV s

400

500

E / mV vs. NHE

400

600

600

E / mV vs. NHE

Fig. 5. Experimental (solid lines) and simulated (dotted lines) CVs for (A) 50 µM FM and 50 µM sulfite in the presence of 10 µM SorT at GC electrode and (B) 50 µM FM and 100 µM sulfite at GC/SorT electrode (5 µL of 117 µM SorT) at different scan rates in 50 mM Tris buffer solution (pH 8).

A comparison of experimental and simulated CVs at various sweep rates (2 to 50 mV s−1) is given in Fig. 5A with SorT, sulfite and FM all freely diffusing in solution (no membrane). Other scan rate dependent simulated voltammograms recorded as a function of sulfite and FM concentrations are given in the Supporting Information (Figs S4-S14). As can be seen from Fig. 5A, at low sulfite concentration (50 μM) and slow sweep rate (2 mV s−1) a highly asymmetric transient CV response is observed which is associated with the depletion of sulfite from the reaction layer (also see Figs. 2A and 2B for examples of this). The wave gradually becomes more symmetrical as the sweep rate increases from 5 to 50 mV s-1 as the diffusion limited FMox/red reaction at the electrode (current proportional to the square root of sweep rate) becomes uncoupled from the FMox-SorTred reaction i.e. the concentration of FMox generated at the 19



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electrode overwhelms the amount of SorTred produced by sulfite oxidation. At higher sulfite concentrations (200-500 µM) and lower FM concentrations (10-25 µM) all CVs become sigmoidal even at relatively rapid sweep rates (50 mV s-1) as the FMox-SorTred reaction becomes dominant and true electrochemical steady state for the FMox/red couple is established. As shown in Fig. 5B (with a membrane present), at low concentrations of sulfite (< 100 µM) the pre-wave at +400 mV is well reproduced by the simulation in addition to the larger diffusional redox wave of FM at +430 mV at 2 mV s-1. As the scan rate increases to 50 mV s-1 the two waves merge and appear as a single slightly asymmetric anodic wave. In summary the same features observed in Figs. 1-3 are well modelled by the simulations including the general desensitization of the current response to increasing sulfite concentration. The effect of increasing the FM concentration in the presence of a saturating amount (500 µM) of sulfite is illustrated in Fig. 6A (all species in solution) and 6B (SorT trapped under a membrane at the electrode surface). It is apparent that the amplitude of the sigmoidal waveform increases with FM concentration. Ideally the plateau current increases with the square root of mediator concentration from theoretical considerations if a true steady state is present.1

A


B

0.2 µΑ


0.1 µΑ

10 µΜ

5 µΜ 10 µΜ

15 µΜ

15 µΜ

20 µΜ

20 µΜ

25 µΜ 25 µΜ

30 µΜ

300

400

500

600

300

E / mV vs. NHE

400

500

E / mV vs. NHE

20


600

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Fig. 6. Experimental (solid lines) and simulated (dotted lines) CVs for varying concentrations of FM (a) in the presence of 500 µM sulfite and 10 µM SorT at GC electrode and (b) in the presence of 500 µM sulfite at GC/SorT electrode (5 µL of 117 µM SorT) [50 mM Tris buffer pH 8 and scan rate: 5 mV s-1].

A single value for the bimolecular outer sphere electron transfer rate constants (k4 = k’4) was used for the FMox (FeIII) - SorTred (MoIV) and FMox (FeIII) - SorTint (MoV) cross reactions (Scheme 2). On the basis of Marcus theory,39, 40 and assuming usual and constant outer sphere reorganizational energies (λ ~ 0.9 eV) and Mo…FM separations in the ‘typical’ range for protein electron transfer (R = 6 to 14 Å),† the rates of oxidation of the MoIV and MoV forms of the enzyme should be within an order of magnitude of each other as long as their MoVI/V and MoV/IV redox potentials are similar (within ca. 200 mV), which is usually the case for other well characterized sulfite oxidising Mo enzymes.41, 42

A Presence of dialysis membrane

B

0.2 µΑ


0.4 µΑ

100 µΜ

25 µΜ

200 µΜ

50 µΜ 400 µΜ

100 µΜ

800 µΜ

200 µΜ

300

400

500

300

600

400

500

600

E / mV vs. NHE

E / mV vs. NHE

Fig. 7. Experimental (solid lines) and simulated (dotted lines) CVs for varying concentrations of sulfite (A) in the presence of 50 µM FM and 10 µM SorT at the GC electrode and (B) in the presence of 50 µM FM at the GC/SorT membrane electrode (5 µL of 117 µM of SorT under the membrane). [50 mM Tris buffer pH 8 and scan rate: 5 mV s-1].

†

The relevant equation is log10ket = 13 – 0.6(R - 3.6) - 3.1 (∆G + λ)2/λ

21



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Fig. 7 illustrates the effect of increasing sulfite concentration in the presence of constant FM (50 µM) and SorT. At low sulfite concentration an asymmetric transient CV was observed due to the depletion of sulfite both in the absence (Fig. 7A) and presence (Fig. 7B) of the dialysis membrane. In Fig. 7A the progression to a true steady state CV is apparent as the sulfite concentration increases. However, in the presence of the dialysis membrane, even at high sulfite concentration (Fig. 7B, 800 µM), the voltammogram still is asymmetric due to mass transport limitations on sulfite reaching the enzyme. These features are well reproduced in the simulation. Table 1. Parameters in Scheme 2 Derived from Electrochemical Simulation (pH 8) Absence of membrane

Presence of membrane

Do(FM) (cm2 s-1)b

4.0 × 10-6

1.2 × 10-7

Do(SO32-) (cm2 s-1)c

1.0 × 10-5

1.0 × 10-6

Do(SorT) (cm2 s-1)d

1.0 × 10-7

1.0 × 10-7

k1 (M-1 s-1)

1.0 × 106

k-1 (s-1)

20

-1

k2 (s )

350

k-2 (s-1)

0.7

k3 (s-1)

3

k-3 (M-1 s-1)

6.0 × 10-3

k4 = k’4 (M-1 s-1)

2.5 × 105

k-4 = k’-4 (M-1 s-1)

1

KM , Sulfite (µM) a

a

370

KM = (k2+k−1)/k1; b Do(FMox) = Do(FMred); c Do(SO32-) = Do(SO42-); d Do(SorTox) = Do(SorTint) = Do(SorTred).

22


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Analysis of Kinetics Parameters. A single set of rate and equilibrium constants defined in Scheme 2 reproduced all our experimental voltammetry undertaken at different sweep rates and various concentrations of sulfite and FM at pH 8. The unique and accurate simultaneous determination of multiple variables is problematic in any simulation due to some parameters having no influence on the simulated CV under certain conditions. Most reactions in Scheme 2 are practically irreversible so the reverse rate constants (k−2, k−3, k−4 and k’−4, in italics in Table 1) are at best upper bounds. The turnover number for sulfite (k2 = 350 s-1) is consistent with the experimental value (338 s-1) published for recombinant SorT from a solution assay with ferricyanide as the electron acceptor.18 The calculated Michaelis constant KM [=(k2+k−1)/k1] (Table 1) is larger than that reported from solution assays17 but this may be linked to the use of a lower than saturating concentration of the artificial electron acceptor (FMox).

Conclusions The mediated electrocatalytic voltammetry of SorT in reaction with its substrate sulfite was demonstrated for the first time using ferrocenium methanol as an artificial electron acceptor. Both transient and sigmoidal voltammograms of the mediator were obtained depending upon substrate concentration and sweep rate. A pH dependence of the catalytic current was demonstrated for SorT consistent with behaviour seen previously in solution assays, although significant loss of activity occurred while the enzyme was kept at pH 10 or above. Digital simulation enabled the kinetics of the enzymatic reaction to be understood as a function of the substrate and mediator concentration at different sweep rates, and a single set of rate constants was obtained that modelled the experimental data. Further, the kinetic parameters obtained from electrochemical simulation were compared in the absence and presence of dialysis membrane and revealed that the diffusion of FM and sulfite to the

23



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electrode surface from the bulk was slowed significantly by the dialysis membrane and this was modelled by lower diffusion coefficients of both the FM mediator and sulfite.

Acknowledgement The Australian Research Council is acknowledged for financial support of this work (DP120101465).

Supporting Information Available. Comparisons of experimental and simulated voltammetry are shown at various concentrations and sweep rates. Control experiments are also shown for non-specific sulfite oxidation and variations in catalytic current due to effects of high pH.

24


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References 1. 2. 3. 4. 5. 6. 7.

8.

9.

10. 11. 12. 13. 14.

15.

16.

17. 18.

Bernhardt, P. V. Enzyme Electrochemistry - Biocatalysis On An Electrode. Aust. J. Chem. 2006, 59, 233-256. Santucci, R.; Picciau, A.; Campanella, L.; Brunori, M. Electrochemistry Of Metalloproteins. Curr. Top. Electrochem. 1994, 3, 313-328. Ruzgas, T.; Csoeregi, E.; Emmneus, J.; Gorton, L.; Marko-Varga, G. Peroxidase-Modified Electrodes: Fundamentals And Application. Anal. Chim. Acta 1996, 330, 123-138. Zuo, P.; Albrecht, T.; Barker, P. D.; Murgida, D. H.; Hildebrandt, P. Interfacial Redox Processes Of Cytochrome B562. Phys. Chem. Chem. Phys. 2009, 11, 7430-7436. Armstrong, F. A.; Wilson, G. S. Recent Developments In Faradaic Bioelectrochemistry. Electrochim. Acta 2000, 45, 2623-2645. Kalimuthu, P.; Leimkühler, S.; Bernhardt, P. V. Catalytic Electrochemistry Of Xanthine Dehydrogenase. J. Phys. Chem. B 2012, 116, 11600-11607. Chen, K.-I.; Mcewan, A. G.; Bernhardt, P. V. Cobalt Hexaamine Mediated Electrocatalytic Voltammetry Of Dimethyl Sulfoxide Reductase: Driving Force Effects On Catalysis. J. Biol. Inorg. Chem. 2011, 16, 227-234. Garguilo, M. G.; Nhan, H.; Proctor, A.; Michael, A. C. Amperometric Sensors For Peroxide, Choline, And Acetylcholine Based On Electron Transfer Between Horseradish Peroxidase And A Redox Polymer. Anal. Chem. 1993, 65, 523-8. Ghach, W.; Etienne, M.; Urbanova, V.; Jorand, F. P. A.; Walcarius, A. Sol-Gel Based 'Artificial' Biofilm From Pseudomonas Fluorescens Using Bovine Heart Cytochrome C As Electron Mediator. Electrochem. Commun. 2014, 38, 71-74. Hille, R.; Hall, J.; Basu, P. The Mononuclear Molybdenum Enzymes. Chem. Rev. 2014, 114, 39634038. Johnson, J. L. Prenatal Diagnosis Of Molybdenum Cofactor Deficiency And Isolated Sulfite Oxidase Deficiency. Prenatal Diagn. 2003, 23, 6-8. Dublin, A. B.; Hald, J. K.; Wootton-Gorges, S. L. Isolated Sulfite Oxidase Deficiency: MR Imaging Features. Am. J. Neuroradiol. 2002, 23, 484-5. Feng, C.; Tollin, G.; Enemark, J. H. Sulfite Oxidizing Enzymes. Biochim. Biophys. Acta, Proteins Proteomics 2007, 1774, 527-539. Eilers, T.; Schwarz, G.; Brinkmann, H.; Witt, C.; Richter, T.; Nieder, J.; Koch, B.; Hille, R.; Hänsch, R.; Mendel, R. R. Identification And Biochemical Characterization Of Arabidopsis Thaliana Sulfite Oxidase: A New Player In Plant Sulfur Metabolism. J. Biol. Chem. 2001, 276, 46989-46994. Schrader, N.; Fischer, K.; Theis, K.; Mendel, R. R.; Schwarz, G.; Kisker, C. The Crystal Structure Of Plant Sulfite Oxidase Provides Insights Into Sulfite Oxidation In Plants And Animals. Structure 2003, 11, 1251-1263. Hänsch, R.; Lang, C.; Riebeseel, E.; Lindigkeit, R.; Gessler, A.; Rennenberg, H.; Mendel, R. R. Plant Sulfite Oxidase As Novel Producer Of H2O2: Combination Of Enzyme Catalysis With A Subsequent Non-Enzymatic Reaction Step. J. Biol. Chem. 2006, 281, 6884-6888. Wilson, J. J.; Kappler, U. Sulfite Oxidation In Sinorhizobium Meliloti. Biochim. Biophys. Acta, Bioenerg. 2009, 1787, 1516-1525. Low, L.; Kilmartin, J. R.; Bernhardt, P. V.; Kappler, U. How Are ‘Atypical’ Sulfite Dehydrogenases Linked To Cell Metabolism? – Interactions Between The Sort Sulfite Deydrogenase And Small Redox Proteins. Front. Microbiol. 2011, 2, Article 58. 25



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

19.

20.

21.

22. 23. 24. 25.

26. 27. 28. 29.

30. 31. 32. 33. 34. 35.

36. 37. 38.

Page 26 of 28

Rajapakshe, A.; Snyder, R. A.; Astashkin, A. V.; Bernardson, P.; Evans, D. J.; Young, C. G.; Evans, D. H.; Enemark, J. H. Insights Into The Nature Of Mo(V) Species In Solution: Modeling Catalytic Cycles For Molybdenum Enzymes. Inorg. Chim. Acta 2009, 362, 4603-4608. Emesh, S.; Rapson, T. D.; Rajapakshe, A.; Kappler, U.; Bernhardt, P. V.; Tollin, G.; Enemark, J. H. Intramolecular Electron Transfer In Sulfite-Oxidizing Enzymes: Elucidating The Role Of A Conserved Active Site Arginine. Biochemistry 2009, 48, 2156-2163. Kappler, U.; Bailey, S. Molecular Basis Of Intramolecular Electron Transfer In Sulfite-Oxidizing Enzymes Is Revealed By High Resolution Structure Of A Heterodimeric Complex Of The Catalytic Molybdopterin Subunit And A C-Type Cytochrome Subunit. J. Biol. Chem. 2005, 280, 2499925007. Kappler, U. Bacterial Sulfite-Oxidizing Enzymes. Biochim. Biophys. Acta, Bioenerg. 2011, 1807, 110. Chen, K.-I.; Mcewan, A. G.; Bernhardt, P. V. Mediated Electrochemistry Of Dimethyl Sulfoxide Reductase From Rhodobacter Capsulatus. J. Biol. Inorg. Chem. 2009, 14, 409-419. Kalimuthu, P.; Leimkühler, S.; Bernhardt, P. V. Low-Potential Amperometric Enzyme Biosensor For Xanthine And Hypoxanthine. Anal. Chem. 2012, 84, 10359-10365. Kalimuthu, P.; Heath, M. D.; Santini, J. M.; Kappler, U.; Bernhardt, P. V. Electrochemically Driven Catalysis Of Rhizobium Sp. NT-26 Arsenite Oxidase With Its Native Electron Acceptor Cytochrome C552. Biochim. Biophys. Acta, Bioenerg. 2014, 1837, 112-120. Kalimuthu, P.; Fischer-Schrader, K.; Schwarz, G.; Bernhardt, P. V. Mediated Electrochemistry Of Nitrate Reductase From Arabidopsis Thaliana. J. Phys. Chem. B 2013, 117, 7569-7577. Broadhead, G. D.; Osgerby, J. M.; Pauson, P. L. Ferrocene Derivatives. V. Ferrocenealdehyde. J. Chem. Soc. 1958, 650-6. Bard, A. J.; Faulkner, L. R. Electrochemical Methods: Fundamentals And Applications. 2001; P Null. Anicet, N.; Bourdillon, C.; Moiroux, J.; Saveant, J.-M. Electron Transfer In Organized Assemblies Of Biomolecules. Step-By-Step Avidin/Biotin Construction And Dynamic Characteristics Of A Spatially Ordered Multilayer Enzyme Electrode. J. Phys. Chem. B 1998, 102, 9844-9849. Situmorang, M.; Hibbert, D. B.; Gooding, J. J.; Barnett, D. A Sulfite Biosensor Fabricated Using Electrodeposited Polytyramine: Application To Wine Analysis. Analyst 1999, 124, 1775-1779. Haladjian, J.; Thierry-Chef, I.; Bianco, P. Permselective-Membrane Pyrolytic Graphite Electrode For The Study Of Microvolumes Of [2Fe-2S] Ferredoxin. Talanta 1996, 43, 1125-1130. Rudolf, M.; Feldberg, S. W. Digisim Version 3.03b. Bioanalytical System, West Lafayette 2004. Leaist, D. G. Moments Analysis Of Restricted Ternary Diffusion: Sodium Sulfite + Sodium Hydroxide + Water. Can. J. Chem. 1985, 63, 2933-2939. Isaac, A.; Davis, J.; Livingstone, C.; Wain, A. J.; Compton, R. G. Electroanalytical Methods For The Determination Of Sulfite In Food And Beverages. Trends Anal. Chem. 2006, 25, 589-598. Kalimuthu, P.; Tkac, J.; Kappler, U.; Davis, J. J.; Bernhardt, P. V. Highly Sensitive And Stable Electrochemical Sulfite Biosensor Incorporating A Bacterial Sulfite Dehydrogenase. Anal. Chem. 2010, 82, 7374-7379. Turner, A. P. F.; Karube, I.; Wilson, G. S. Biosensors: Fundamentals And Applications. Oxford University Press: 1987. Kalimuthu, P.; Leimkühler, S.; Bernhardt, P. V. Xanthine Dehydrogenase Electrocatalysis: Autocatalysis And Novel Activity. J. Phys. Chem. B 2011, 115, 2655-2662. Renkin, E. M. Filtration, Diffusion And Molecular Sieving Through Porous Cellulose Membranes. J. Gen. Physiol. 1954, 38, 225-243. 26


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39. 40. 41.

42.

Moser, C. C.; Keske, J. M.; Warncke, K.; Farid, R. S.; Dutton, P. L. Nature Of Biological Electron Transfer. Nature 1992, 355, 796-802. Page, C. C.; Moser, C. C.; Chen, X.; Dutton, P. L. Natural Engineering Principles Of Electron Tunnelling In Biological Oxidation-Reduction. Nature 1999, 402, 47-52. Rapson, T. D.; Kappler, U.; Hanson, G. R.; Bernhardt, P. V. Short Circuiting A Sulfite Oxidising Enzyme With Direct Electrochemistry: Active Site Substitutions And Their Effect On Catalysis And Electron Transfer. Biochim. Biophys. Acta, Bioenerg. 2011, 1807, 108-118. Spence, J. T.; Kipke, C. A.; Enemark, J. H.; Sunde, R. A. Stoichiometry Of Electron Uptake And The Effect Of Anions And Ph On The Molybdenum And Heme Reduction Potentials Of Sulfite Oxidase. Inorg. Chem. 1991, 30, 3011-15.

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Table of Contents Graphic

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Catalytic Voltammetry of the Molybdoenzyme Sulfite Dehydrogenase

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