Chitosan-Promoted Direct Electrochemistry of Human Sulfite Oxidase

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Chitosan-Promoted Direct Electrochemistry of Human Sulfite Oxidase Palraj Kalimuthu, Abdel A. Belaidi, Guenter Schwarz, and Paul Vincent Bernhardt J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b06712 • Publication Date (Web): 05 Sep 2017 Downloaded from http://pubs.acs.org on September 9, 2017

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The Journal of Physical Chemistry

Chitosan-Promoted Direct Electrochemistry of Human Sulfite Oxidase Palraj Kalimuthu,a Abdel A. Belaidi,b,c Guenter Schwarzb and Paul V. Bernhardta,* a

School 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 c

The Florey Institute of Neuroscience and Mental Health, University of Melbourne, Victoria, 3052, Australia

E-mail: [email protected]

Abstract Direct electrochemistry of human sulfite oxidase (HSO) has been achieved on carboxylateterminated self-assembled monolayers cast on a Au working electrode in the presence of the promoter chitosan. The modified electrode facilitates a well-defined non-turnover redox response from the heme cofactor (FeIII/II) in 750 mM Tris, MOPS and bicine buffer solutions. The formal redox potential of the non-turnover response varies slightly depending on the nature of the thiol monolayer on the Au electrode. Upon addition of sulfite to the cell a pronounced catalytic current from HSO-facilitated sulfite oxidation is observed. The measured catalytic rate constant (kcat) is around 0.2 s-1 (compared with 26 s-1 obtained from solution assays), which indicates that interaction of the enzyme with the electrode lowers overall catalysis although native behaviour is retained in terms of substrate concentration dependence, pH dependence and inhibition effects. In contrast, no catalytic activity is observed when HSO is confined to amine-terminated thiol monolayers although well-defined non-catalytic responses from the heme cofactor are still observed. These differences are linked to flexibility of HSO, which can switch between active and inactive conformations and also competitive ion exchange processes at the electrode surface involving the enzyme and substrate.

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Introduction Human sulfite oxidase (HSO) is an essential ~110 kD homodimeric heme-molybdoenzyme located within the inter-membrane space of mitochondria.1 HSO catalyses the oxidation of sulfite to chemically inert sulfate in the final step of cysteine degradation. Dysfunction in HSO activity is a fatal condition if not treated immediately due to severe neurological damage that is caused by toxic sulfite. Lack of HSO activity may be due to molybdenum cofactor (Moco) deficiency whereby mutations in proteins essential for Moco biosynthesis (MOCS1, MOCS2 and GPHN) lead to acute deficiency of sulfite oxidase (as well as the other three Mo-dependent enzymes in humans).2-4 Moco deficiency can now be treated by early (neonatal) supplementation of a vital Moco precursor.5 Isolated sulfite oxidase deficiency is caused by mutations in the gene encoding HSO and result in an enzyme that is also ineffective in sulfite oxidation.3, 6-8 The crystal structure of the highly homologous (68% identical) chicken liver SO (CSO) reveals two subunits with the Mo active site and heme cofactors connected by a flexible peptide loop.8 The Nterminal domain contains a b-type heme cofactor while the larger C-terminal domain harbours the active site bearing the Moco ligand (a bidentate dithiolene chelator) coordinated to Mo in addition to a cysteine ligand, an axial oxido ligand and an equatorial water based ligand that is exchanged with the substrate and whose protonation state varies with the Mo oxidation state. In the crystal structure of CSO the Mo and heme cofactors are separated by 32 Å and catalytically essential intramolecular electron transfer (IET) is unfeasible in this conformation. Kinetic studies have examined how the flexible peptide chain connecting the two subunits allows the two cofactors to reorient to a closer distance (estimated to be ~12 Å but still unknown), thereby facilitating an IET and catalysis.9-11 Molecular dynamics simulations have also been applied to this question.12

2


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Scheme 1. Electron transfer and conformational lability in HSO (monomer shown). The colour scheme is green=fully oxidized (MoVI and FeIII; orange=fully reduced (MoIV and FeII) and pink=half-reduced (MoV). IET refers to intramolecular electron transfer and the black line represents the flexible linker connecting the Mo and heme domains.

The electron transfer sequence is summarized in cartoon form in Scheme 1 where the abovementioned conformational dynamics of the flexible linker (represented by the solid black line) has been included for some of the HSO forms; in principle all forms are capable of adopting an ‘active’ (cofactors in proximity) and ‘inactive’ (cofactors separated) conformation. The role of the ferric heme cofactor is to relay electrons between the Mo active site and the natural electron acceptor cytochrome c. This must happen in sequence as the ferric heme can only accept one electron from the two electron reduced MoIV centre. The two consecutive single electron oxidations of the heme are preceded by intramolecular IET where the Mo and Fe oxidation states equilibrate and this can only occur when the Mo and heme cofactors are in proximity as shown. Overall the reaction stoichiometry is given by eqn 1. 3



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HSO

2+ SO23 +H2 O+2(cyt. c)𝑜𝑥 → SO4 +2H +2(cyt. c)𝑟𝑒𝑑

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(1)

It has been well established in both vertebrate13-21 and bacterial sulfite dehydrogenases22, 23 that a mediator (either synthetic or natural) can facilitate electron transfer between the enzyme and the electrode. However, there are restrictions on the type of mediator that can be employed. Sulfite may be oxidized non-specifically at an electrode so the redox potential of the mediator must be well below this (ca. +600 mV vs NHE at pH 8). Moreover, the charge of the mediator plays an important role in favouring (or opposing) non-covalent interactions with surface amino acids in vicinity of the enzyme cofactors.21 The quest for direct electrochemical responses of oxidoreductase enzymes, without a redox mediator, is driven by potential applications in biofuel cells, biomedical devices and electrocatalysis.24-27 Direct (unmediated) electrochemistry of sulfite oxidizing enzymes offers the advantage of driving catalysis at very low (effectively zero) overpotential with no interference from nonspecific oxidation of competing species at the electrode surface. Due to their size and inaccessibility of the redox active centers buried within the enzyme, heterogeneous electron transfer remains a challenge. There have been various approaches to this problem across the family of vertebrate19, 28-32 and bacterial33-35 sulfite oxidising enzymes. Direct catalytic voltammetry of CSO has been achieved by two independent groups adopting distinctly different approaches. In both cases a non-turnover response from the heme cofactor enabled the amount of electroactive enzyme to be quantified. However, not all electroactive CSO molecules were catalytically competent. When CSO was adsorbed on a self-assembled monolayer (SAM) comprising amine and hydroxyl terminated alkyl chains covalently bound to Au via thiolate groups the electrochemical turn-over number (kcat = 20 s-1)19 was about 20% of that obtained from solution assay.36 When CSO was adsorbed on pyrolytic graphite electrode the percentage of catalytically competent 4


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enzyme was even lower (~4%).28 It was suggested that the fraction of catalytically inactive CSO molecules on the electrode are those trapped in a conformation where the Mo and heme cofactors are far apart (Scheme 1). The direct electrochemistry of HSO has been carried out on various chemically modified electrode surfaces with different degrees of sensitivity.29, 31, 37, 38 One of the best performing examples was reported by Weidinger and co-workers who employed a Ag electrode modified with a mixed SAM comprising 8-mercaptooctanamine and 6-mercaptohexanol.29 Both non-turnover (heme) and turnover (catalytic) voltammetric responses were found. Interestingly the catalytic activity of the electrode-adsorbed enzyme was found to be sensitive to buffer concentration. The conclusion was that increasing the buffer concentration enhances the mobility of the SAM-adsorbed HSO through competitive non-covalent interactions and the enzyme is better able to function without restrictions of electrode adsorption. Recently, we reported the mediated catalytic voltammetry of HSO and heme-free HSO with positively and negatively charged synthetic electron acceptors20, 21 and found that mediator charge plays a vital role in catalytic activity. Positively charged hexaamineiron(III) complexes effectively interact with the more negatively charged heme domain through electrostatic attraction. On the other hand, the negatively charged mediator [Fe(CN)6]3- interacts more favourably with the more positively charged Moco domain of HSO. Nevertheless the negatively charged heme domain of HSO still is capable of donating electrons to the negatively charged electron acceptor [Fe(CN)6]3- but at a slower rate.21 In the present study, we have investigated the direct electrochemistry of HSO on SAM modified Au electrodes terminated with different functional groups; both positively and negatively charged. The catalytic activity is examined as a function of pH, concentration of buffer and temperature. Moreover, the kinetic parameters towards sulfite catalytic oxidation are explored and compared with previous reports where possible. The introduction of the biopolymer chitosan is important in providing a stable electrochemical response from HSO. 5



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Experimental Materials Holo and heme free HSOs were expressed and purified in E. coli TP1000 as described.39 Sodium sulfite,

3-mercaptopropionic

acid,

3-mercaptosuccinic

acid,

4-mercaptobenzoic

acid,

11-

mercaptoundecanoic acid, cysteamine and 5-(4-aminophenyl)-1,3,4-oxadiazole-2-thiol were purchased from Aldrich and were used as received. Chitosan from shrimp shells, (≥75% deacetylated) was obtained from Sigma-Aldrich. All other reagents used were of analytical grade purity and used without any further purification. Tris acetate, 3-(N-morpholino)propanesulfonic acid (MOPS) and bicine buffers were used for single buffer experiments. For pH-dependent experiment, a mixture of buffers 150 mM 2-(Nmorpholino)ethanesulfonic acid (MES) buffer, 150 mM Bis-Tris, 150 mM Tris buffer, 150 mM Ncyclohexyl-2-aminoethanesulfonic acid (CHES) buffer and 150 mM N-cyclohexyl-3-aminopropanesulfonic acid (CAPS) buffer were 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).

Electrochemical Measurements and Electrode Cleaning Cyclic voltammetry (CV) experiments were carried out with a BAS 100B/W electrochemical workstation. A three-electrode system was employed comprising a gold disk working electrode, a Pt wire counter, and an Ag/AgCl reference electrode (+196 mV vs NHE) all manufactured by BAS. Potentials are cited versus NHE. Experiments were carried out on solutions that had been purged with Ar gas for at least 30 min and an Ar blanket was maintained during the measurement. The Au working electrode was mechanically, chemically, and electrochemically cleaned and polished according to a published procedure.40 The thiol monolayers were prepared by immersing a clean polycrystalline Au electrode into 5 mM ethanolic solutions of either 3-mercaptopropionic acid, 3-mercaptosuccinic acid, 4mercaptobenzoic acid, cysteamine or 5-(4-aminophenyl)-1,3,4-oxadiazole-2-thiol for 3 h and 116


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mercaptoundecanoic acid for 24 h. The thiolate-modified electrodes were subsequently washed thoroughly with ethanol and water to remove any unbound thiols then dried in a nitrogen atmosphere before enzyme immobilization.

Enzyme Electrode Preparation A mixture of 3 µL 50 µM HSO (or heme free HSO) and 0.25 % chitosan (2.0 µL) (ca. 14 mM glucosamine monomers) was pipetted onto the conducting surface of an inverted, freshly prepared thiolate-modified Au working electrode and this was allowed to dry to a film at 4°C. To prevent protein loss the enzyme modified electrode surface was carefully covered with a perm-selective dialysis membrane (molecular weight cutoff 3.5 kDa), 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 enzyme modified electrode was stored at 4°C in 50 mM Tris buffer (pH 8.0) when not in use. The enzyme and chitosan are both confined to a thin layer beneath the membrane while sulfite and buffer were able to diffuse and equilibrate either side of the dialysis membrane. The electro-active surface area of the Au electrode (A) was determined from cyclic voltammetry of 1 mM ferrocene methanol41 in 0.1 M KCl solution at different sweep rates using the Randles-Sevcik equation (eqn 2).42 1/2

𝑖p = (2.69 × 105 )𝑛3/2 𝐴𝐷𝑜 𝐶𝑜 1/2

(2)

The standard diffusion coefficient (Do) of ferrocene methanol is 6.7 × 10-6 cm2 s-1,43 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).

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The surface coverage (Γ, mol cm-2) of electroactive HSO confined to the modified electrode was calculated from the anodic voltammetry peak (eqn 3),44 where ip is the peak current of the non-turnover response, F is the Faraday constant (96,485 C mol−1) and the other symbols have their usual meaning. 𝑖p =

𝑛2 𝐹 2  𝛤𝐴 4𝑅𝑇

(3)

The limiting catalytic peak current (ilim) was measured at +200 mV vs NHE, which is within a potential independent plateau beyond the sigmoidal wave. The variation of ilim as a function of sulfite concentration was fit to Michaelis–Menten kinetics (eqn 4) yielding KM,app (the apparent Michaelis constant) and imax (the effective electrochemical turnover number). 𝑖lim =

𝑖max [SO2− 3 ] 𝐾M,app + [SO2− 3 ]

(4)

The catalytic turnover number (kcat) was calculated from eqn 5.19 This applies only when mass transport is not rate limiting ([SO32-] » KM,app), thus ilim = imax (from eqn 4) 𝑘cat =

𝑖max 𝑛𝐴𝐹

(5)

The pH dependence of the catalytic current was modeled with eqn 636 which is applicable for an enzyme that is deactivated by either deprotonation of an acid at high pH (pKa1) or protonation of a base at lower pH (pKa2) and iopt is the maximum activity at the pH optimum. 𝑖opt

𝑖max (𝑝𝐻) = 1+10(𝑝𝐻−𝑝𝐾𝑎1 )+10(𝑝𝐾𝑎2 −𝑝𝐻)

8


(6)

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Results and Discussion Non-turnover Voltammetry Previous studies of CSO and HSO have found that the negatively charged heme domain of HSO has an affinity for positively charged functional groups at the terminus of SAMs cast on Au working electrodes. Nevertheless, we found a clear non-turnover redox response from the heme cofactor of HSO on a negatively charged, carboxylate-terminated SAM modified electrode.

A

B

a

b a

0.05

b

I 

0

I 

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0

–0.1

–0.05 0

0

200

200

E/ mV vs. NHE

E/ mV vs. NHE

Figure 1. CVs obtained for HSO (a) 1st cycle and (b) 6th cycle at a Au/MPA electrode in (A) the absence and (B) the presence of chitosan (750 mM Tris buffer, pH 8.5) and scan rate 5 mV s-1. Figure 1A shows the non-turnover cyclic voltammetry (CV) response of HSO on a 3mercaptopropionate (MPA) modified Au electrode. A reversible redox response centered at +73 mV vs NHE is apparent (Figure 1A, curve a) with a peak-to-peak separation of 75 mV in the first sweep. This is attributed to the one electron FeIII/II redox response for the b-type heme in HSO,29 which has been reported to lie in the range +54 to +62 mV vs NHE from optical spectroelectrochemistry experiments.9, 45

9



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However, the CV redox response degrades in subsequent sweeps as can be seen for the sixth sweep (Figure 1A, curve b and also supporting information Figure S1).

A possible cause of the unstable signal is that the short chain MPA forms a sparse and less ordered monolayer where exposed areas on the Au electrode become fouled by irreversibly adsorbed and denatured enzyme resulting in surface passivation. A similar observation was reported for CSO on short chain thiols comprising cysteamine and 2-mercaptoethanol modified Au electrodes.19 The presence of the dialysis membrane should enable a reversible association/dissociation between HSO and the electrode as the enzyme cannot escape to the bulk solution, but the observed loss of current is irreversible under these conditions which points to electrode fouling.

To solve this problem we introduced the biopolymer chitosan to improve the affinity between HSO and the Au/MPA electrode. The commercial sample of chitosan (75% deacteylated chitin) is heterogeneous but molecular weights well in excess of 100 kDa are typical (comprising more than 500 glucosamine monomers).46, 47 Given the stoichiometry of electrode preparation (ca. 150 pmol HSO vs 28 nmol glucosamine monomers; ratio of ~180 glucosamine monomer:1 HSO) the number of HSO proteins under the membrane is in excess of chitosan polymers by a factor of at least 3 if a conservative average chitosan molecular weight of 100 kDa is assumed. Therefore, even though HSO is in excess of chitosan there is enough chitosan present to facilitate and stabilize interactions between the Au/MPA surface and HSO.

The result was a stable redox response for HSO mixed with chitosan on the Au/MPA electrode (Figure 1B). A quasi-reversible non-turnover redox response at +38 mV with a peak to peak separation of 75 mV was observed which is a similar potential reported for HSO at positively charged polyelectrolyteentrapped quantum dots37 and gold nanoparticle modified electrodes,30 as well as the above-mentioned 10


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spectroelectrochemical data for HSO.9, 45 Unlike the data in Figure 1A, the CV response is stable over multiple cycles (Figure 1B, curves a and b). At the experimental pH of 8.5 chitosan is essentially charge neutral as nearly all its glucosamine monomers will be in their free base form; the chitosan protonation constants lie in the range 6.5 < pKa < 7.48 Therefore, H-bonding must be the dominant non-covalent stabilizing force in this case between the SAM terminal –COO- groups, HSO and chitosan.

As a control, the CV of heme-free HSO under the same conditions (Supporting Figure, S2) does not show any Mo-based redox response. The Mo cofactor is seemingly inaccessible for interfacial electron transfer under these conditions and this was not pursued further. The MoVI/V redox potential of HSO has been determined to be +42 mV vs NHE (pH 7.5) from a combination of spectroelectrochemistry and laser flash photolysis experiments.10 The highly homologous CSO has been characterized by EPR potentiometry and the redox potentials are MoVI/V +70 and MoV/IV -90 mV vs NHE at pH 7.49 The absence of any redox response from heme-free HSO is also indirect support for assignment of the above redox peak at +38 mV in holo-HSO to the heme cofactor.

We extended the study of holo-HSO to include other carboxylate- and amino-functionalized aliphatic and aromatic SAMs comprising 3-mercaptosuccinate (MSA), 4-mercaptobenzoate (MBA), 11mercaptoundecanoate, cysteamine (2-mercaptoethanamine) and 5-(4-aminophenyl)-1,3,4-oxadiazole-2thiol. As found for the Au/MPA electrode system, chitosan was essential otherwise HSO electroactivity was lost after a few cycles. Well defined non-turnover redox responses were observed for HSO at +52 mV and +55 mV at Au/MSA and Au/MBA modified electrodes, respectively. No redox response was observed for HSO on a Au/11-mercaptoundecanoate electrode, which is similar to a previous report for CSO at a Au/11-mercaptoundecanoate electrode.19 However, the length of the alkyl chain alone is not the cause of this behaviour as SAMs of similar length but bearing different head groups (–NH3+ and –OH) were suitable for direct electron transfer with CSO.19 11



0.05

A

0.2

e

B b

a

I 

I 

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0

a 0

–0.05

c d

–0.2 0

200

0

E/ mV vs. NHE

200

E/ mV vs. NHE

Figure 2. CVs (scan rate 5 mV s-1, pH 8.0) obtained for the non-turnover redox response of HSO at a Au/MPA/chitosan electrode (A) with different Tris buffer concentrations (a) 100, (b) 250, (c) 500, (d) 750 and (e) 1000 mM and (B) in 750 mM (a) TAPS, (b) Tris, (c) MOPS and (d) bicine. The different colours are for emphasis.

The non-turnover redox response of HSO was very sensitive to both buffer concentration and composition. In Figure 2A the initially weak heme response at the Au/MPA/chitosan/HSO electrode in 100 mM Tris buffer increases significantly in magnitude with concentration of buffer (from 100 to 1000 mM). Varying the buffer but maintaining the same pH also had a marked influence on the CV response. In Figure 2B the four CVs are all from the same Au/MPA/chitosan/HSO electrode placed in different buffer solutions all at a concentration of 750 mM and pH 8.0. For TAPS (curve a) a weak response is observed which increases in magnitude when the electrode was placed in 750 mM Tris buffer solution (curve b). This response increases again when MOPS and bicine are employed (curves c and d). At pH 8 TAPS, MOPS and bicine are all mixtures of a zwitterion and an anion while Tris is a mixture of cationic and neutral molecules. The pKa values all fall in a similar range (7.5 < pKa < 8.5). None of these features appears responsible for the differences seen in Figure 2B.

12


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Chitosan is also a vital part of the matrix. As mentioned above at pH 8, chitosan is essentially charge neutral yet in its absence the CV response is unstable. Therefore, H-bonding interactions mediated by chitosan, not electrostatic forces, appear dominant in stabilizing the Au/MPA-HSO interaction. TAPS and Tris share the same tris-hydroxymethylmethanamine core and, like chitosan, are replete with H-bond donors, comprising three primary alcohols and a protonated amine. Bicine and MOPS are tertiary amines and also possess fewer H-bond donors. We speculate that Tris and TAPS are sufficiently potent H-bond competitors with chitosan and disrupt the electroactive HSO/chitosan matrix while MOPS and bicine are less competitive.

However, as illustrated in Figure 2A, even the relatively poorly promoting buffer Tris elicits a good CV response when its concentration is raised. Although this seems at odds with the previous discussion, in this case the higher ionic strength has the effect of mobilizing HSO and labilising the dynamic HSO-electrode interaction. Similar empirical observations have been made in the past with HSO as a function of ionic strength.29-31 Above all, these HSO-electrode attractive forces are very weak and easily disrupted. The heme non-turnover redox response is lost upon removal of the dialysis membrane in 750 mM Tris buffer solution (Supporting information, Figure S3), which further indicates that HSO does not physically adsorb on the electrode but instead is continually associating and dissociating with the SAM head groups.

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The Journal of Physical Chemistry 1.2

B

1

A

y = 0.2208x - 0.545 R2 = 0.9824

1.0

0.8

I / A

I 

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y = 0.0216x - 0.0243 R2 = 0.9996

0.6

0

0.4

a 0.2

e 0.0

0

200

0

E/ mV vs. NHE

10

20

30

40

50

(Scan rate)1/2 or Scan rate in mV.s-1

60

Figure 3. (A) CVs obtained for a Au/MPA/chitosan/HSO electrode in 750 mM bicine buffer (pH 8.5) at different scan rates (a) 10, (b) 20, (c) 30, (d) 40 and (e) 50 mV s-1 and (B) plot of anodic current vs scan rate (squares) or square root of scan rate (circles). The colours are included to discriminate the different scan rates. The sweep rate dependence of the current response for HSO on a Au/MPA/chitosan electrode (750 mM bicine, pH 8.5) is shown in Figure 3. Discrimination between the response for a surfaceconfined redox couple and one limited by diffusion can normally be distinguished by either a linear increase in current with sweep rate (adsorbed, Figure 3B squares) or a linear increase in current with the square root of sweep rate (diffusional, Figure 3B circles).42 In this case the sweep rate range was limited by rather sluggish heterogeneous electron transfer, which resulted in peak broadening to the point that accurate subtraction of the baseline charging current was not possible above a sweep rate of about 50 mV s-1. As can be seen in Figure 3B it is not possible to discriminate between the two models in this case. The enzyme is confined to a small volume under the membrane so in principle diffusion is still possible. However, given the size of HSO (110 kDa) and its consequently slow rate of diffusion, the response in Figure 3A is most likely due to a surface confined enzyme at the SAM interface. The effect of varying the SAM chain length whilst keeping the –COO- head group constant (and buffer, 750 mM Tris, pH 8.5) is illustrated in the Supporting Information (Figs S4-S6). Similar redox responses from the heme cofactor

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were seen at the MSA and MBA-modified Au electrodes although the current response was somewhat higher on the MSA-modified electrode. The surface coverage (, pmol cm-2) of electroactive enzyme in each case was calculated for the each SAM-modified Au electrode from eqn 3. The slope of the linear current/sweep rate plots (Figure 3B) equates to

𝒏𝟐 𝑭𝟐 𝜞𝑨 𝟒𝑹𝑻

where n=1 and the electrode area A = 0.055 cm2. These calculations are

qualified by a rather large uncertainty (ca. 10 nA) in the anodic current, which was measured relative to a large capacitance current. These calculations (from Figs S4-S6) lead to approximate values of  that lie in the range 10-40 pmol cm-2 (Table 1) which is higher than the surface coverage obtained for HSO at positively charged polyelectrolyte-entrapped quantum dot modified electrodes (4.2 pmol cm-2)37 and also for CSO adsorbed on hydroxyl and amine-terminated mixed SAMs cast on a Au electrode (1.3 pmol cm-2)19. The HSO heme response at a Au/MPA electrode showed little variation across the range 4.5 < pH < 10 (Supporting Information, Fig. S7). The current response was essentially the same and there was only a small shift in redox potential over this pH range which shows that no proton transfer involving the ligands associated with heme cofactor occur over this pH range during electron transfer.

Catalytic Voltammetry As mentioned above well-defined non-catalytic redox responses are seen for HSO on MPA, MSA and MBA modified Au electrodes with chitosan as a promoter (Figure 4A-C, curve a), but not 11mercaptoundecanoate (Figure 4D, curve a). Upon addition of 4 mM sulfite to the electrochemical cell the transient response of the heme cofactor is replaced by a sigmoidal waveform accompanied by an increase in current (Figure 4A-C, curve b). The forward and backward sweeps are identical (apart from differences due to capacitive current) and the cathodic current vanishes. The observed sigmoidal 15



waveform is characteristic of an electrochemical steady state where HSO is reoxidised at the electrode at the same rate as it is reduced by sulfite. The catalytic redox potential coincides with the non-turnover redox potential of the heme cofactor which illustrates that catalysis occurs at essentially the lowest possible applied potential. The catalytic response of HSO was also investigated in the absence of chitosan on the Au/MPA electrode.

A

B

b

0.1

b

I 

I 

0.1

a

0

a

0

0

200

0

E/ mV vs. NHE 0.1

C

200

E/ mV vs. NHE

D

b

b a

a

I 

I 

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

Page 16 of 31

0

0

–0.05 0

200

–200

E/ mV vs. NHE

0

200

E/ mV vs. NHE

Figure 4. CVs obtained in the (a) absence and (b) presence of 4 mM of sulfite at (A) Au/MPA/chitosan/HSO, (B) Au/MSA/chitosan/HSO, (c) Au/MBA/chitosan/HSO and (d) Au/11mercaptoundecanoate/chitosan/HSO in 750 mM Tris buffer (pH 8.0) at a scan rate of 5 mV s-1.

16


Page 17 of 31

Again the catalytic sigmoidal wave increased significantly upon addition of 4 mM sulfite (supporting information, Figure S8). However, as already illustrated (Figure 1), chitosan stabilizes the HSO-electrode interaction so for practical purposes chitosan was retained for all experiments. As expected from the lack of a heme response, no catalytic current was observed for HSO on a Au/11mercaptoundecanoate electrode in the presence of sulfite (Figure 4D curve b). Ferapontova et al. found similar results with CSO using this particular SAM cast on a Au electrode.19 Non-specific sulfite oxidation (in the absence of HSO) is negligible within this potential window (-200 to +300 mV vs NHE, supporting information, Figure S9).

A

0.2

b

0.2

B

b

0.1

I 

a

I 

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


a 0

0 –0.1

0

200

0

E/ mV vs. NHE

200

E/ mV vs. NHE

Figure 5. CVs obtained for the (a) absence and (b) presence of 4 mM of sulfite at (A) Au/cysteamine/chitosan/HSO and (B) Au/5-(4-aminophenyl)-1,3,4-oxadiazole-2-thiol/chitosan/HSO in 750 mM Tris buffer at a scan rate of 5 mV s-1. The electrochemistry of HSO was also investigated on amino-terminated SAMs cast on a Au electrode (Figure 5). 5-(4’-Aminophenyl)-1,3,4-oxadiazole-2-thiol is an aromatic amine and so the –NH2 group should be in its free base form above pH 5. The amino group of cysteamine is much more basic (pKa > 10)50 so this SAM head group will be mostly protonated within the pH range of these experiments. 17



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Page 18 of 31

Non-turnover redox responses of HSO were observed at +52 mV and +60 mV on cysteamine and 5-(4aminophenyl)-1,3,4-oxadiazole-2-thiol monolayers, respectively (Figures 5A and 5B, curves a). Upon addition of 4 mM sulfite to the cell the non-turnover heme response at both electrodes slowly diminishes and eventually is lost altogether (Figure 5A and 5B, curves b and supporting information Figure S10). No catalytic current is ever observed. A similar observation has been reported for CSO on cysteamine SAM modified electrodes.19 It likely in this case that sulfite displaces HSO from the SAM surface through a competitive ion-exchange process and results in irreversible modification of the SAM as the non-turnover responses seen in Figures 5A and 5B did not return on replacing the electrolyte solution with fresh buffer. This is a very unusual observation as previous studies have shown favourable electroactivity of HSO on electrodes chemically modified with positively charged ammonium head groups.29, 30, 32, 37, 38 The conventional belief is that the positive charges attract the negatively charged surface amino acids in proximity to the heme domain of HSO. For example, catalytic activity was achieved on positively charged thin films such as polyethyleneimine (PEI) capped CdS nanoparticles,37 PEI-capped gold nanoparticles30 and aminopropyltriethoxysilane modified ITO electrodes.38 Table 1. Electrochemical parameters for HSO on different modified Au electrodes (750 mM Tris, pH 8.5). Self-assembled Monolayer Eo (mV vs NHE) kcat (s-1)a icat (nA)b  (pmol)c MPA

+38

0.33

56.7

16

MSA

+53

0.24

90.7

35

MBA

+63

0.17

36.1

20

a

Calculated from eqn 5; b The catalytic current measured in the presence of 4 mM sulfite at a scan rate of 5 mV s−1; c calculated From eqn 3. 18


Page 19 of 31

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Given the surface coverage of electroactive HSO calculated from the non-turnover heme response, the catalytic rate constant (kcat) for HSO-sulfite turnover was calculated for the carboxylate functionalized monolayers and these values were found to be essentially the same (~0.2 s-1) given the uncertainty in the value of  (Table 1). In other words the observed catalytic current is directly proportional to the surface coverage of HSO but not markedly affected by the type of –COO- terminated SAM. The electrochemical kcat values are lower than that reported from solution assays of HSO (26 s-1),7 which illustrates that most of the electrode-confined HSO molecules are catalytically inactive despite being able to exchange electrons with the heme. Vertebrate sulfite oxidizing enzymes are distinct from bacterial and plant derived analogs, which lack the flexible peptide loop connecting the Mo and heme cofactors.51 That is, only vertebrate sulfite oxidizing enzymes possess conformational lability of the kind shown in Scheme 1, which modulates the rate of IET between the Mo and heme domains. Given that interfacial electron transfer is apparent from the heme non-turnover response, the low catalytic activity points to an inability of some HSO molecules to achieve a conformation that facilitates IET (Scheme 1). However, the fact that the catalytic response is purely sigmoidal (and not a superposition of a transient and steady state waveform) suggests that all electroactive heme cofactors are in some way involved in mediating electron transfer between the catalytically active HSO enzymes and the electrode.

Sulfite Concentration Dependence The electrochemical response of the HSO modified electrode was studied as a function of sulfite concentration (Tris, pH 8.5) and the data are shown in Figure 6. As shown before a well-defined noncatalytic redox response appears at +38 mV for HSO on a Au/MPA/chitosan electrode in the absence of sulfite (Figure 6A, curve a). A classical sigmoidal catalytic waveform emerges upon addition of 50 µM sulfite to the buffer solution (Figure 6A, curve b) and this wave increases gradually for subsequent additions of sulfite up to 3200 µM (curves c-h). From the sulfite concentration dependence of the 19



catalytic current (Figure 6B) we calculate an apparent Michaelis constant KM,app= 497 µM (eqn 5). This value is more than an order of magnitude greater than reported for HSO from solution assays at this pH (11 μM).52 Similarly high electrochemical KM values have been reported for HSO on positively charged mixed monolayer modified Ag electrode (KM,app = 170 µM) in 750 mM Tris buffer solution.29

100 90

A

B

80 70

i

I / nA

0.1

I 

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Page 20 of 31

a

60

KM = 497 

50 40 30

0

20 10 0

0

200

0

E/ mV vs. NHE

1000

2000

3000

Concentration (M)

4000

Figure 6. (A) CVs obtained for the increasing concentration of sulfite (a) 0, (b) 100, (c) 200, (d) 400, (e) 800, (f) 1600, (g) 2400, (h) 3200 and (i) 4000 µM at Au/MPA/chitosan/HSO in 750 mM Tris buffer solution at a scan rate of 2 mV s-1 (B) Michaelis-Menten plot (eqn 4) for the baseline subtracted electrocatalytic oxidation current at +100 mV vs NHE.

The cause for the observed high KM,app value in the present study is mass transport limitations imposed by the dialysis membrane covering the electrode. Sulfite must cross the membrane from the bulk solution and this in effect slows the rate of formation of the Michaelis complex (the so called onrate). It is noted that essentially the same value (KM,app = 512 µM) was reported for electrochemically driven HSO catalysis mediated by the complex [Fe(tacn)2]3+ (tacn = 1,4,7-triazacyclononane) on a glassy carbon electrode (covered by a membrane).20 This reaffirms that mass transport limitations effectively

20


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desensitize the system and extend the linear current response to sulfite well beyond the natural

100

80

60

I / nA

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


40

20

0 5

6

7

8

9

10

11

pH Michaelis constant.

Figure 7. pH dependence of the maximum catalytic current at a Au/MPA/chitosan/HSO electrode with 4 mM sulfite in 750 mM mixed buffer solutions at a scan rate of 5 mV s-1. The solid curve is obtained from a fit to the experimental data using eqn 6 (pKa1 10.2 and pKa2 7.2).

pH Dependence The pH dependence of catalytic sulfite oxidation at the Au/MPA/chitosan/HSO electrode was explored in the range of 5.5 < pH < 11 in 750 mM mixed buffer solution (Figure 7) and the experimental voltammograms are shown in supporting information Figure S11. The catalytic current exhibits a pH optimum of 8.5 which is the same as that recently reported for HSO with iron complexes as electron transfer mediators (pH 8.5).20 A bell shaped profile was modelled with eqn 6 which enabled two pKa values to be determined (7.2 and 10.2); the lower value defining the protonation constant of a base that switches off catalysis and the higher one being the deprotonation constant of an acid that also is 21



essential for catalysis. These observed pKa values are consistent with our recent report for mediated catalytic voltammetry of HSO with iron complexes (7.0 and 9.8)20 and also match those determined from solution assays of HSO with natural electron acceptor cytochrome c (7.3 and 10.2).52 The relative insensitivity of the heme response to pH (Fig. S7) rules out a change in the concentration of electroactive HSO as a function of pH.

-15.0

A o

Activation energy 29.4 kJ mol-1

-15.4

Activation enegry = 29.4 kJ mol

ln (i) (i in A)

0.2

B

-15.2

40 C o 35 C

I 

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-15.6

o

30 C o 25 C o 20 C o 15 C o 10 C o 5C

-1

-15.8 -16.0 -16.2

0

-16.4 -16.6 -16.8 -3

0

200

-3

-3

-3

-3

-3

-3

-3

-3

-3

3.1x10 3.2x10 3.3x10 3.3x10 3.4x10 3.4x10 3.5x10 3.5x10 3.6x10 3.6x10

1/T (T in K)

E/ mV vs. NHE

Figure 8. (A) CVs obtained for Au/MPA/chitosan/HSO with 4 mM sulfite at different temperatures (a) 5, (b) 10, (c) 15, (d) 20, (e) 25, (f) 30, (g) 35 and (e) 40 oC in 750 mM Tris buffer (pH 8.5) at a scan rate of 5 mV s-1. (B) Arrhenius plot of the natural logarithm of maximum catalytic current versus the reciprocal of absolute temperature (K).

Temperature Dependence The catalytic activity of the Au/MPA/chitosan/HSO electrode was also investigated at varying temperatures by keeping the concentration of enzyme, substrate and buffer constant. As shown in Figure 8A the catalytic current increases markedly upon increasing the temperature from 5 to 40 oC. The activation energy was estimated from an Arrhenius plot (Figure 8B, lnimax versus the T-1) yielding a value

22


Page 23 of 31

of ΔH‡ of 29.4 kJ mol-1. This value is comparable with that reported for CSO at a polyaniline modified electrode (23.2 kJ mol−1).53

0.1

d

B

A e

c

I 

0.5

I 

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


a

b

0

a 0 0

200

0

E/ mV vs. NHE

200

E/ mV vs. NHE

Figure 9. CVs obtained for 4 mM sulfite at the Au/MPA/chitosan/HSO electrode in (A) different Tris buffer concentration at pH 8.5 (a) 100, (b) 250, (c) 500, (d) 750 and (e) 1000 mM and (B) in 750 mM (a) TAPS (b) Tris (c) MOPS and (d) bicine buffer (pH 8.0 in each case). The scan rate is 5 mV s-1

Buffer Influence As mentioned earlier, the buffer concentration and composition has a large influence on the magnitude of the non-turnover response of the heme (Figure 2A and 2B). Not surprisingly this has a flow-on effect to the catalytic response. As illustrated in Figure 9A the catalytic sulfite oxidation current significantly increases with Tris buffer concentration (pH 8.0) from 100 mM to 1 M. Assuming that the amount of surface confined HSO remains the same during this experiment this points to an enhancement of the apparent kcat value. Given that conformational mobility of HSO is correlated with activity, the higher buffer concentration may lead to a competitive adsorption process between the 23



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Page 24 of 31

buffer and HSO for the electrode surface and weakening the HSO-SAM interaction frees up the enzyme for conformational movement. This was also seen in the direct catalytic voltammetry of HSO at PEI capped Au nanoparticle modified electrodes.30 Molecular dynamics calculations12 have concurred with this hypothesis and found that at low ionic strength HSO is (too) strongly adsorbed to the SAM through its heme domain, which inhibits the domain motion required for IET. As already demonstrated, the buffer itself also has a major effect on the non-turnover heme signal (Figure 2B). We also analyzed the catalytic response of HSO in various buffer solutions while keeping buffer concentration (750 mM) and pH constant. Figure 9B illustrates the catalytic response of HSO on a Au/MPA electrode with 4 mM sulfite in different Tris buffer solutions (pH 8.0). A weak catalytic wave is seen in TAPS buffer solution (curve a). In contrast, a well pronounced classical sigmoidal wave is found when the enzyme modified electrode is in Tris buffer solution (curve b). The catalytic response is further increased in MOPS buffer (curve c). Again, the catalytic current is increased markedly in bicine buffer solution (curve d). However, no significant catalytic response observed for HSO if the bicine buffer concentration was lowered to 100 mM (data not shown). These results mirror the data in Figure 2B which showed the same qualitative trend in the non-turnover current as a function of buffer. This again indicates that the catalytic response is affected in a major way both by concentration and composition of the buffer and that these phenomena can easily override effects such as pH and substrate concentration.

24


Page 25 of 31

a b

0.4

I 

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c

0.2

0

0

200

E/ mV vs. NHE Figure 10. CVs obtained for the increasing concentration of phosphate ions (a) 0, (b) 0.5 M and (c) 1 M in the presence of 4 mM sulfite at Au/MPA/chitosan/HSO electrode in 750 mM Tris buffer (pH 8) at a scan rate of 5 mV s-1.

Inhibition Effects It has been reported that phosphate is a reversible inhibitor for CSO54 and HSO32 and that the IET rate decreases significantly from 1500 to less than 100 s-1 in its presence.55 The inhibition effect of phosphate was investigated here on the catalytic response of HSO at a Au/MPA electrode and the data are shown in Figure 10. A typical catalytic response is observed in the presence of 4 mM sulfite in 750 mM Tris buffer (pH 8.0) (curve a). When the phosphate concentration is raised to 0.5 M and then 1 M the catalytic current decreases consistent with the known phosphate inhibition of HSO. Electrostatic

25



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Page 26 of 31

interactions between the heme domain and Mo domain are weakened by the binding of phosphate ions near the Mo active site which leads a significant decrease of IET and loss of catalytic activity.

Conclusions We have demonstrated native catalytic activity of HSO using a working electrode as the only electron transfer partner. Previous studies have focussed on positively charged, ammonium-terminated self-assembled monolayers to achieve direct electron transfer with the enzyme. Herein we have presented a new approach that utilises negatively charged (–COO- terminated) self-assembled monolayers which are effective in facilitating direct electron transfer with the heme domain. The biopolymer chitosan plays an important role in stabilising the HSO-electrode interaction although direct electron transfer is still possible in its absence. A well-defined reversible ferric/ferrous heme response was found for a variety of SAMs terminated by negatively charged, positively charged and charge neutral functional groups. Upon addition of sulfite a catalytic current is observed at the redox potential of the heme cofactor for the carboxylate-terminated SAMs cast on an Au electrode only. The catalytic activity of HSO is sensitive to pH, with an optimum around pH 8.5 and the enzyme is inhibited reversibly by phosphate in line with solution assays.

Acknowledgements PVB acknowledges financial support from the Australian Research Council (Discovery Grant DP150103345). SUPPORTING INFORMATION AVAILABLE Non-turnover, catalytic and control (no enzyme) cyclic voltammetry for HSO at different SAM modified electrodes, pH dependence and also the influence of chitosan and dialysis membrane on stability of the redox response. 26


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15.

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23. 24. 25. 26.

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A

b

0.1

sulfite

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


a

no sulfite

0

0

200

E/ mV vs. NHE

1


Chitosan-Promoted Direct Electrochemistry of Human Sulfite Oxidase

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