Mediated Electrochemistry of Nitrate Reductase from Arabidopsis

Article pubs.acs.org/JPCB

Mediated Electrochemistry of Nitrate Reductase from Arabidopsis thaliana Palraj Kalimuthu,† Katrin Fischer-Schrader,‡ Günter Schwarz,‡ and Paul V. Bernhardt*,† †

School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, 4072, Australia Institute of Biochemistry, Department of Chemistry & Center for Molecular Medicine, Cologne University, Zülpicherstr. 47, 50674 Köln, Germany

‡

S Supporting Information *

ABSTRACT: Herein we report the mediated electrocatalytic voltammetry of the plant molybdoenzyme nitrate reductase (NR) from Arabidopsis thaliana using the established truncated molybdenum-heme fragment at a glassy carbon (GC) electrode. Methyl viologen (MV), benzyl viologen (BV), and anthraquinone-2-sulfonic acid (AQ) are employed as effective artificial electron transfer partners for NR, differing in redox potential over a range of about 220 mV and delivering different reductive driving forces to the enzyme. Nitrate is reduced at the Mo active site of NR, yielding the oxidized form of the enzyme, which is reactivated by the electroreduced form of the mediator. Digital simulation was performed using a single set of enzyme dependent parameters for all catalytic voltammetry obtained under different sweep rates and various substrate or mediator concentrations. The kinetic constants from digital simulation provide new insight into the kinetics of the NR catalytic mechanism.

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In the catalytic reaction, nitrate binds to the (reduced) MoIV form displacing a hydroxido/aqua ligand, forming the Michaelis complex. Upon inner sphere oxidation of the Mo center to MoVI by nitrate, an oxido ligand (originating from a nitrate Oatom) is formed and nitrite is released, as illustrated in Scheme 1. After completion of the oxidative half reaction, the active MoIV form is regenerated by NAD(P)H which binds adjacent to the FAD cofactor (Figure 2).9 Electron transfer from FAD to the Mo active site occurs via the heme relay. Artificial electron donors such as reduced viologens can also regenerate the active enzyme9,11 as in conventional chemical assays where an external reductant such as dithionite is required to sustain the catalytic reaction and continually recycle the donor. Alternatively, electrochemical reduction of the mediator removes the need for a chemical reductant as an external current takes its place.12 When electrochemical simulation of the reaction cycle (Scheme 1) is carried out, the kinetics of various steps in the catalytic cycle may be quantified; many of these reactions are too rapid to be measured by conventional stopped flow kinetic methods.13−15 Only a few direct (unmediated) electrochemical reports of a NR enzyme have appeared, and all have involved bacterial systems from the DMSO reductase family (Nar, Nap, and Nas).16−21 Even some atypical electrochemical behavior has

INTRODUCTION

There are several classes of nitrate reducing Mo enzymes, which each catalyze the two-electron reduction of nitrate to nitrite. In bacteria, the dissimilatory nitrate reductases (Nar and Nap) and assimilatory (Nas) enzymes all belong to the socalled DMSO reductase family (Figure 1, left) due to their similar active site structure comprising two bidentate molybdopterin ligands, an oxido/hydroxido ligand, and an amino acid residue (Cys or Asp).1 The eukaryotic assimilatory nitrate reductases found in plants, fungi, and algae are also Mo enzymes yet bear completely different structures. In particular, the active site comprises a five-coordinate Mo ion coordinated to a single molybdopterin ligand (Figure 1, right), a cysteine residue, and two terminal water based ligands (oxido/hydroxido/aqua), placing these enzymes in the sulfite oxidase family.1 Nitrate reductase (NR) enzymes from this family catalyze the reduction of nitrate using pyridine nucleotides as the reductant.2−4 The monomeric ca. 100 kDa component of NR comprises three domains bearing a cofactor: the Mo active site, a heme (cytochrome b5), and an FAD domain (with an NADH binding site).5−8 A cofactor free dimerization domain is also present, as shown in Figure 2, and the active enzyme is a homodimer.9,10 The central heme domain bridges both C and N terminal domains via two protease-sensitive solvent exposed loops, namely, hinge 1 and hinge 2. © XXXX American Chemical Society

Received: April 25, 2013 Revised: May 31, 2013

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dx.doi.org/10.1021/jp404076w | J. Phys. Chem. B XXXX, XXX, XXX−XXX

The Journal of Physical Chemistry B

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Figure 1. The variety of active sites found in nitrate reducing molybdoenzymes.

Figure 2. Schematic representation of the full length and truncated plant NR lacking the FAD domain.

Direct electrochemistry of eukaryotic NR enzymes has not been successful to date, and the problems may stem from the sheer size of the homodimer (ca. 200 kDa)5,23 and the fact that the redox active cofactors are deeply buried inside the protein hindering heterogeneous electron transfer. To overcome this issue, low molecular weight redox compounds may be employed as redox mediating cosubstrates, which facilitate electron transfer between electrode and enzyme. These mediated electrochemical studies have involved bacterial and fungal Mo enzymes (Escherichia coli,24,25 yeast,26 Aspergillus,27−31 Parracoccus,32 Cupriavidus,22 and Pseudomonas species33). Electrochemically driven catalytic studies on plant NR are yet to appear in the literature. Organic aromatic and heterocyclic compounds including methyl viologen, azure A, safranin T, phenosafranin, bromophenol blue, janus green B, indigo-5-5′-disulfonic acid, and neutral red have been investigated as electron transfer partners for various NR enzymes.25,29−31,34,35 Viologens possess low redox potentials, so they are an ideal choice in an artificially reconstituted nitrate reducing system. Generally, these redox dyes are coimmobilized with NR by different methods

Scheme 1. Catalytic Mechanism at the Plant Nitrate Reductase Active Site

been reported in NR enzymes due to deleterious effects of physisorption on an electrode surface.22

Figure 3. Chemical structures and redox potentials of the mediators employed in this paper (pH 7, potential vs NHE). B



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Scheme 2. Mediated Electrochemically Driven Catalysis of NR

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including adsorption, cross-linking, entrapment, electro-polymerization, and covalent binding on different substrates including gold, glassy carbon, and edge plane pyrolytic graphite electrodes.28,33,36 In recent years, we have employed both mediated and direct catalytic electrochemistry with a variety of other molybdoenzymes, some of which have potential applications in bioanalytical sensing.11,13,15,37,38 The redox potentials of the Mo, heme, and FAD cofactors of plant NR (from spinach and corn leaf) have been reported previously and collectively fall in the range −300 to +15 mV vs NHE (FAD < heme < MoV/V/IV).3,39,40 The truncated NR, lacking the FAD cofactor, has been characterized in previous studies11 and utilized exclusively in this study (see Figure 2). In the present study, we report the electrocatalytic voltammetry of recombinant plant NR from Arabidopsis thaliana at a glassy carbon electrode with the mediating substrates methyl viologen (MV), benzyl viologen (BV), and anthraquinone-2-sulfonic acid (AQ), which have redox potentials (Figure 3) that are all lower than the enzyme active site. Further, we have performed electrochemical simulation to explore the rate and equilibrium constants of the NR catalytic reaction and also the reaction with its artificial electron partners by modeling the catalytic voltammograms of truncated NR over a range of mediator and substrate concentrations. This work provides new insight into the kinetics of the NR catalytic mechanism not accessible from steady state or stopped flow kinetics studies.

EXPERIMENTAL SECTION

Materials. Arabidopsis thaliana nitrate reductase was purified from a heterogeneous expression system in E. coli as previously described.11 Sodium nitrate, methyl viologen, benzyl viologen, and anthraquione-2-sulfonic acid were purchased from Aldrich and were used as received. All other reagents used were of analytical grade purity and used without any further purification. All solutions were prepared with ultrapure water (resistivity 18.2 MΩ·cm) from a Millipore Milli-Q system. Bis− Tris−acetate (50 mM) in the presence of 50 mM KCl as inert electrolyte was used for experiments buffered at pH 7. Electrochemical Measurements and Electrode Cleaning. Cyclic voltammetry (CV) was measured with a Bioanalytical Systems BAS 100B/W electrochemical workstation. A three-electrode system was employed comprising a glassy carbon (GC) disk working electrode, a Pt wire counter, and an Ag/AgCl reference electrode (+196 mV vs NHE), all supplied by Bioanalytical Systems. Potentials are cited versus NHE. Experiments were carried on solutions that had been purged with Ar for 30 min at 298 K. The GC electrode was polished with 0.50 and 0.05 μm alumina slurry and then rinsed thoroughly with water. The electrode was then sonicated in water for 5 min to remove any adsorbed alumina particles and dried in a nitrogen atmosphere. The sweep rate was in the range 5−50 mV s−1. Aliquots of concentrated sodium nitrate solution were added by pipet between each scan for substrate concentration dependent experiments (under an Ar atmosphere) to give the desired bulk concentration. C



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Enzyme Electrode Preparation. A 3 μL droplet of NR (36 μM) in 50 mM buffer (50 mM Bis−Tris−acetate (pH 7.0), 50 mM KCl, 5 mM magnesium acetate, and 1 mM CaCl2) was pipetted onto the conducting surface of an inverted, freshly prepared GC working electrode, and this was allowed to dry to a film at 4 °C. To prevent protein loss, the electrode surface was carefully covered with a perm-selective dialysis membrane (molecular weight 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 the internal membrane solution. The resulting modified electrode was stored at 4 °C in 50 mM Bis− Tris buffer (pH 7.0) when not in use. The enzyme was confined to a thin layer beneath the membrane, while nitrate/ nitrite and mediator were able to diffuse across the membrane. Electrochemical Simulation. To simulate the experimental cyclic voltammograms (CVs), the DigiSim (version 3.03b) program was used.42 The electro-active surface area of the Au electrode (A = 0.055 cm2) was determined by CV analysis of a 1 mM ferrocene methanol solution (0.1 M KCl) at different sweep rates using the Randles−Sevcik equation (eq 1).43 The diffusion coefficient (Do) of ferrocene methanol is 6.7 × 10−6 cm2 s−1,44 ip is the measured current maximum, the number of electrons is n = 1, the concentration of analyte is Co = 10−6 mol cm−3, and ν is the sweep rate (V s−1). i p = (2.69 × 105)n3/2ADo1/2Coν1/2

mechanism is employed to model the overall reaction kinetics (Scheme 2). In the reductive half reaction, the mediator reduces the enzyme active site (MoVI in its resting state) in an outer sphere electron transfer reaction in consecutive oneelectron steps (k4) via an intermediate (MoV) form NRint. Internal electron transfer (heme → Mo) is assumed to be rapid and not rate limiting, and this has been measured in separate experiments.11,23 In our model system using truncated NR, the heme is the initial site of reduction. Nitrate binds to the reduced active site (k1). In the oxidative half reaction, the MoIV converts the coordinated nitrato ligand to nitrite (k2),23 and the nitrite product dissociates from the active site (k3). Here, we have carried out NR catalytic voltammetry with three mediators MV and BV and AQ. Therefore, a MoV intermediate is possible if each mediator donates a single electron and in fact it is likely to proceed in this way given the fact that the heme is the more accessible cofactor and can only accept a single electron at a time. Catalytic Voltammetry. An example of the mediated catalytic voltammetry of NR is illustrated in Figure 4. Although

(1)

The double-layer capacitance was determined to be 12 μF. Semi-infinite diffusion was assumed, and all pre-equilibration reactions were disabled. The apparent redox potential of mediators was determined from experiments in the absence of NR and nitrate. The diffusion coefficients of mediators were obtained by simulation of their cyclic voltammetry at varying sweep rates in the absence of substrate and enzyme to give values of 1 × 10−5, 2 × 10−5, and 3 × 10−5 cm2 s−1 for MV, BV, and AQ, respectively. The diffusion coefficients for NR and nitrate were taken to be 1 × 10−8 and 1 × 10−5 cm2 s−1. All diffusion coefficients were kept constant thereafter. The heterogeneous rate constants (ks) were determined by simulating the sweep rate dependence of the anodic peak to cathodic peak separation of mediators (in the absence of NR) and then held constant during the simulations of different sweep rate and substrate and mediator concentration dependent catalytic voltammetry. The ks values were 2 × 10−2 cm s−1 (BV), 8 × 10−2 cm s−1 (MV), and 9 × 10−4 cm s−1 (AQ). The only parameters that were allowed to vary during the simulation were the homogeneous rate and equilibrium constants shown in Scheme 2.

Figure 4. CVs obtained for AQ (25 μM) in the absence (a) and presence (b) of 800 μM nitrate at the GC/NR electrode in 50 mM Bis−Tris buffer containing 50 mM KCl (pH 7) at a sweep rate of 5 mV s−1.

truncated NR has two electro-active centers (heme and Mo), no “non-turnover” redox responses were observed from either cofactor in the absence of nitrate at the GC working electrode (data not shown). However, the GC/NR electrode shows a well-defined redox wave at ca. −250 mV vs NHE in the presence of 25 μM AQ (pH 7) with a peak-to-peak separation of 80 mV (curve a). The waveform and sweep rate dependence is consistent with a quasi-reversible, diffusion controlled redox response of the mediator. The electrode reaction involves an overall twoelectron and two-proton reduction of anthraquinone-2-sulfonic acid (AQ) to dihydroanthraquinone-2-sulfonic acid (AQH2) and subsequent reoxidation of AQH2 to AQ.45 Upon addition of 800 μM nitrate to the electrochemical cell, a well-defined sigmoidal waveform appears and the limiting cathodic peak increases by an order of magnitude (curve b). No anodic wave was present in this case. This voltammetry is characteristic of a catalytic homogeneous reaction coupled to heterogeneous electron transfer (EC′ mechanism)43 where nitrate is reduced enzymatically, yielding the oxidized form of NR, which is reduced again by electro-generated AQH2. As will be

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RESULTS AND DISCUSSION Electrocatalytic Mechanism of NR. Full-length NR is a complex homodimeric enzyme in which each subunit possesses three cofactors (FAD, heme, and Mo) bound to distinct and independently folded domains.6−8 As mentioned above, we have utilized a truncated form of NR in this work, which lacks the FAD cofactor (Figure 2) but is fully active when electrons are provided to the heme cofactor directly by a mediator such as methyl viologen.11 We have assumed that the catalytic reaction follows Michaelis−Menten kinetics and it is associated with the substrate binding (k1/k−1), turnover (k2/k−2), and product release (k3/k−3). A simplified double substrate “ping-pong” D



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homogeneous reaction with NRox) at the same rate that it is generated at the electrode surface. Nitrate Reductase−Mediator Reaction. The NR− mediator reaction was examined with the varying AQ, BV, and MV concentrations in the presence of a high (constant) nitrate concentration. Figure 6A shows the situation for BV in the presence of 1 mM nitrate at a sweep rate of 5 mV s−1. At 10 μM BV, a sigmoidal voltammogram is observed, indicative of an electrochemical steady state where both the forward and backward sweeps are almost the same. At higher BV concentrations (20 and 30 μM), the waveform of the voltammogram becomes asymmetric due to an excess of BVred being produced at the electrode which overwhelms the now limiting amount of NRox formed in the nitrate reduction step; the BV concentration is no longer at steady state. Figure 6B displays similar experiments but this time with increasing MV concentrations (2, 4, and 6 μM). An unusual feature is seen in the lowest MV concentration where the curves cross over on the forward and reverse sweeps. The phenomenon is a consequence of the MV mediator diffusion (to and from the enzyme/electrode) limiting the current. This is only seen at very low concentrations of MV and at slow sweep rates. None of the other mediators exhibited this feature, and this is due to MVred reacting more rapidly with NRox than the two higher potential mediators. As the bulk MV concentration increases, the curves no longer cross over, although there is still a noticeable depletion as the cathodic current drops off as the potential is lowered. It should be emphasized that the MV concentrations in Figure 6B are much lower than BV in Figure 6A or AQ in Figure 4. Thus, in this case, it is mediator depletion (at low concentration) that leads to this drop in current (in an analogous way to nitrate depletion; see Figure 4 at low nitrate concentration). Electrochemical Simulation. In recent years, we have employed digital simulation for a better understanding of the mechanism of mediated enzyme electrochemical reactions.13−15 The objective of the simulation is to deduce the rate constants defined in Scheme 2 that can reproduce all voltammetric features over a range of sweep rates and substrate or mediator concentrations. Figures 4−6 already illustrate the diversity of CV profiles that can be observed. Sweep Rate Dependence. The voltammetric sweep rate is a significant variable to elucidate the kinetics of electrochemical processes coupled with chemical reactions, and the DigiSim program42 enables the same set of rate constants to be

demonstrated later, AQH2 acts as a single electron donor to NR. Nitrate Reductase−Nitrate Reaction. The enzyme− substrate reaction was investigated by varying nitrate concentration while maintaining a constant concentration of mediator and enzyme. As an example, the CV of NR in the presence of 25 μM AQ at a sweep rate of 5 mV s−1 is shown in Figure 5. At low nitrate concentrations (25 μM), the CV takes

Figure 5. CVs obtained for varying nitrate concentrations (25, 200, and 800 μM) in the presence of 25 μM AQ at the GC/NR electrode in 50 mM Bis−Tris buffer containing 50 mM KCl as electrolyte, pH 7 at a sweep rate of 5 mV s−1.

the form of an irreversible transient wave with a pronounced cathodic peak but no corresponding anodic peak. This feature is due to depletion of nitrate at the electrode surface, which is consumed by NR at a rate that exceeds its replenishment by diffusion from the bulk solution. Depletion of nitrate attenuates the rate at which AQ is regenerated, thus leading to a drop in the AQ cathodic current. As the nitrate concentration is raised (Figure 5, 200 μM), the wave increases in magnitude but retains a similar asymmetric shape. Finally, at very high concentrations of nitrate (Figure 5, 800 μM), a sigmoidal wave emerges where the concentration of nitrate within the reaction layer is constant (no depletion) during the sweep and the forward and reverse sweeps are virtually identical. The voltammogram is indicative of an electrochemical steady state; i.e., AQH2 is consumed (by

Figure 6. CVs obtained for varying (A) BV and (B) MV concentrations in the presence of 1 mM nitrate at the GC/NR electrode in 50 mM Bis− Tris buffer containing 50 mM KCl as electrolyte, pH 7 at a sweep rate of 5 mV s−1. E



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Figure 7. Experimental (solid lines) and simulated (broken lines) sweep rate dependent CVs obtained for (A) 25 μM MV and 800 μM nitrate, (B) 25 μM BV and 1600 μM nitrate, and (C) 25 μM AQ and 100 μM nitrate at the GC/NR electrode in 50 mM Bis−Tris buffer containing 50 mM KCl (pH 7).

optimized for CVs measured across a range of sweep rates but under an identical set of concentrations (enzyme, mediator, and substrate). When the concentrations of mediators and nitrate are varied, then ideally the same parameters will reproduce the CVs measured under those situations as well. In Figure 7, the experimental and simulated CVs are shown for MV, BV, and AQ (25 μM), each at different concentrations of nitrate, as a function of sweep rate. All other sweep rate dependent simulated voltammograms recorded as a function of various mediator and substrate concentrations are given in the Supporting Information. Each set (Figure 7A, B, and C) illustrates a different feature. In Figure 7A, an initial transient irreversible CV at slow sweep rate is due to MVred depletion where oxidation by NRox outruns the rate at which it is generated at the electrode. As the sweep rate increases, the wave takes on a more sigmoidal appearance, as there is now sufficient MVred being produced to sustain a steady state with the coupled NR/nitrate reaction. In Figure 7B, nearly all of the CVs are sigmoidal and importantly the ones measured at 20 and 50 mV s−1 are almost identical (except for a slightly greater charging current at 50 mV s−1). This is characteristic of a situation where the concentration of MVred at the electrode surface is at steady state with its electrochemical formation exactly matching its consumption by NRox. For this to occur, the NRred−nitrate reaction rate must be fast and constant, which is achieved at high (saturating) nitrate concentrations, and this rate at least matches the rate that MVred is produced at the electrode at these sweep rates. In other words, it is the substrate turnover step (rate k2) that is controlling the kinetics in this situation. The substrate binding rate (k1) was determined accurately from CVs at lower nitrate concentrations, and trial values less than ca. 105 M−1 s−1 led to a significantly worse fit. The product dissociation rate k3 value also has a significant influence on the CV if allowed to drop below its optimal value in Table 1. The apparent turnover number (k2) is correlated with the concentration of active enzyme and variations in this number from one enzyme preparation to the next. In the current study, the same enzyme sample was used which eliminated activity differences and all electrodes and samples were prepared afresh. Apart from intrinsic variations in activity, variations in the volume of solution in contact with the electrode (under the membrane) will lead to different enzyme concentrations as the absolute amount of enzyme added in each case was the same

Table 1. Kinetic Parameters in Scheme 2 Obtained from Electrochemical Simulation MV

BV

AQ

k1 (M−1 s−1)

1.0 × 105

1.0 × 105

1.0 × 105

k−1 (s−1) k2 (s−1) k−2 (s−1) k3 (s−1) k−3 (M−1 s−1) KM,app,nitrate (mM) E° (mV vs NHE) k4 (M−1 s−1) k−4 (M−1 s−1)

166 75 0.2 50 0.5 2.2

166 75 0.2 50 0.5 2.2

166 75 0.2 50 0.5 2.2

−434

−324

−230

3.5 × 103 2.9

1.6 × 103 1.3

1.0 × 104 10

mediator independent

mediator dependent

but again our method is quite reproducible and the same set of enzyme dependent parameters could be used throughout. Finally, each step was effectively irreversible (kn ≫ k−n), so the rates of the reverse reactions in Table 1 (k−n, n = 1−4) are nominal and had no significant effect on the simulation. Mediator Concentration Dependence. The kinetic parameters obtained from the sweep rate dependence in the previous section also reproduced the variations in voltammetry as a function of mediator concentration. The increasing concentration of mediators such as MV (2−6 μM), BV (10− 20 μM), and AQ (10−20 μM) in the presence of 1 mM nitrate at a sweep rate of 10 mV s−1 is represented in Figure 8. The fit is very good in each case, and even the unusual curve crossing phenomenon seen in the case of MV is reproduced (Figure 8A). Further, we found that the catalytic current saturated at 10 and 30 μM of MV and BV, respectively, in the presence of 1 mM nitrate. Substrate Concentration Dependence. The same set of parameters in Table 1 was used to simulate the substrate concentration-dependent catalytic voltammograms. Figure 9 displays the cathodic current response of the GC/NR electrode as a function of nitrate concentration in the presence of 25 μM of each mediator. The catalytic current was essentially linear up to nitrate concentrations of 2.4, 1.6, and 0.8 mM for MV, BV, and AQ, respectively but approached an asymptote thereafter. It is apparent that the catalytic current attains saturation quickly when the reductive driving force is small but extends to very F



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Figure 8. Experimental (solid lines) and simulated (broken lines) CVs obtained for varying mediator concentration in the presence of 1 mM nitrate: (A) MV, (B) BV, and (C) AQ at the GC/NR electrode in 50 mM Bis−Tris buffer containing 50 mM KCl (pH 7) at a sweep rate of 5 mV s−1.

Figure 9. Experimental (solid lines) and simulated (broken lines) CVs obtained for varying nitrate concentration in the presence of 25 μM mediators: (A) MV, (B) BV, and (C) AQ at the GC/NR electrode in 50 mM Bis−Tris buffer containing 50 mM KCl (pH 7) at a sweep rate of 10 mV s−1.

determination of multiple parameters is problematic in that some parameters will have no effect depending on the concentrations of the reactants. We were careful to ensure that the lower bounds of each rate constant were reached so that their values were meaningful. The second order rate constants k1 and k4 were most accurately obtained from simulations at low nitrate and mediator concentrations, respectively. The turnover number (k2) obtained in the simulations is consistent with experimental values published for NR by solution assays (33 s−1).41 It is interesting that all three mediators BVred, MVred, and AQH2 behaved as one-electron donors. The two viologens are common one-electron donors in their reduced radical monocation form. In contrast, AQH2 is generally a twoelectron and two-proton reductant at neutral pH46 because the semiquinone (AQH) is unstable with respect to disproportionation (EAQ/AQH < EAQH/AQH2). Nevertheless, the simulations clearly show that AQH2 (generated at the electrode by twoelectron reduction) acts as a single electron donor to NR in the same way as the obligate one-electron donors MVred and BVred. In this case, disproportionation of AQH (the product of the outer sphere electron transfer with NR) may occur rapidly after its formation. Simulations attempted with a two-electron mediated electron transfer from AQH2 led to poor fits and very different voltammetric waveforms. The slope, or steepness, of the rising part of the sigmoidal waveform is characteristic of the number

high substrate concentrations when the driving force is very large (MV). We reported similar phenomena before the mediated electrochemistry of DMSO reductase where the linear response of the enzyme may be extended well beyond its KM value.14 The calculated KM,app values in Table 1 are based on Michaelis−Menten kinetics (KM = (k2+k−1)/k1) and are about 10 times larger than determined from solution assays (164 μM).11,41 It should be remembered that in a double substrate (pingpong) mechanism such as this the maximum catalytic current will only be observed at saturating concentrations of both nitrate and mediator. Although the catalytic current does indeed saturate at high nitrate concentrations, the corresponding experiment with the mediator cannot be carried out. As the mediator concentration rises, the enzyme is ultimately unable to maintain a steady state of the reduced mediator, and the sigmoidal steady state voltammetric response is replaced by the transient response of the (excess) mediator which then continues to increase linearly with concentration characteristic of a transient voltammetry response.43 Examples of this may be seen in Figures 6A and 8B. Analysis of Kinetics Parameters. The rate and equilibrium constants in Table 1 and defined in Scheme 2 reproduced all our experimental voltammetry carried out at different sweep rates and various concentrations of nitrate and mediators. Importantly, the same set of mediator-independent parameters (k1−k3) was used in all systems. The accurate G



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of electrons transferred in the activating step.47 This single electron transfer stoichiometry is consistent with the heme cofactor, the more accessible cofactor, only being able to interact with single electron donors by shuttling electrons one at a time to the Mo cofactor. The heme redox potential of the truncated form of NR is −187 mV vs NHE.11 The slightly faster MV reduction of NR compared with BV is consistent with Marcus theory (log ket ∝ −ΔG2),48 with the lower redox potential of MV generating a greater driving force. However, the AQ/AQH/AQH2 system is different and the k4 value cannot be explained simply on the basis of Marcus theory and the observed redox potential. Spontaneous disproportionation of two molecules of AQH in the vicinity of the enzyme will generate an extra molecule of AQH2 which may reduce an additional NR enzyme in its vicinity. This cannot occur in MV or BV which are stable against disproportionation. There is also the possibility that the Mo active site is reduced directly by AQH2, as has been demonstrated for a heme-deficient variant of NR in its reduction by bromphenol blue.11

(5) Redinbaugh, M. G.; Campbell, W. H. Quaternary Structure and Composition of Squash NADH:Nitrate Reductase. J. Biol. Chem. 1985, 260, 3380−3385. (6) Crawford, N. M.; Smith, M.; Bellissimo, D.; Davis, R. W. Sequence and Nitrate Regulation of the Arabidopsis thaliana mRNA Encoding Nitrate Reductase, a Metalloflavoprotein with Three Functional Domains. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 5006− 5010. (7) Vaucheret, H.; Kronenberger, J.; Rouzé, P.; Caboche, M. Complete Nucleotide Sequence of the Two Homeologous Tobacco Nitrate Reductase Genes. Plant Mol. Biol. 1989, 12, 597−600. (8) Gowri, G.; Campbell, W. H. cDNA Clones for Corn Leaf NADH:Nitrate Reductase and Chloroplast NAD(P):Glyceraldehyde3-Phosphate Dehydrogenase: Characterization of the Clones and Analysis of the Expression of the Genes in Leaves as Influenced by Nitrate in the Light and Dark. Plant Physiol. 1989, 90, 792−798. (9) Fischer, K.; Barbier, G. G.; Hecht, H.-J.; Mendel, R. R.; Campbell, W. H.; Schwarz, G. Structural Basis of Eukaryotic Nitrate Reduction: Crystal Structures of the Nitrate Reductase Active Site. Plant Cell 2005, 17, 1167−1179. (10) Lu, G.; Campbell, W. H.; Schneider, G.; Lindqvist, Y. Crystal Structure of the FAD-Containing Fragment of Corn Nitrate Reductase at 2.5 A Resolution: Relationship to Other Flavoprotein Reductases. Structure 1994, 2, 809−821. (11) Lambeck, I. C.; Fischer-Schrader, K.; Niks, D.; Roeper, J.; Chi, J.-C.; Hille, R.; Schwarz, G. Molecular Mechanism of 14-3-3 ProteinMediated Inhibition of Plant Nitrate Reductase. J. Biol. Chem. 2012, 287, 4562−4571. (12) Bernhardt, P. V. Enzyme Electrochemistry - Biocatalysis on an Electrode. Aust. J. Chem. 2006, 59, 233−256. (13) Chen, K.-I.; McEwan, A.; Bernhardt, P. Mediated Electrochemistry of Dimethyl Sulfoxide Reductase from Rhodobacter capsulatus. J. Biol. Inorg. Chem. 2009, 14, 409−419. (14) 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. (15) Kalimuthu, P.; Leimkühler, S.; Bernhardt, P. V. Catalytic Electrochemistry of Xanthine Dehydrogenase. J. Phys. Chem. B 2012, 116, 11600−11607. (16) Anderson, L. J.; Richardson, D. J.; Butt, J. N. Using Direct Electrochemistry to Probe Rate Limiting Events during Nitrate Reductase Turnover. Faraday Discuss. 2000, 116, 155−169. (17) Anderson, L. J.; Richardson, D. J.; Butt, J. N. Catalytic Protein Film Voltammetry from a Respiratory Nitrate Reductase Provides Evidence for Complex Electrochemical Modulation of Enzyme Activity. Biochemistry 2001, 40, 11294−11307. (18) Elliott, S. J.; Hoke, K. R.; Heffron, K.; Palak, M.; Rothery, R. A.; Weiner, J. H.; Armstrong, F. A. Voltammetric Studies of the Catalytic Mechanism of the Respiratory Nitrate Reductase from Escherichia coli: How Nitrate Reduction and Inhibition Depend on the Oxidation State of the Active Site. Biochemistry 2004, 43, 799−807. (19) Frangioni, B.; Arnoux, P.; Sabaty, M.; Pignol, D.; Bertrand, P.; Guigliarelli, B.; Leger, C. Rhodobacter sphaeroides Respiratory Nitrate Reductase, the Kinetics of Substrate Binding Favors Intramolecular Electron Transfer. J. Am. Chem. Soc. 2004, 126, 1328−1329. (20) Fourmond, V.; Burlat, B.; Dementin, S.; Sabaty, M.; Arnoux, P.; Etienne, E.; Guigliarelli, B.; Bertrand, P.; Pignol, D.; Leger, C. Dependence of Catalytic Activity on Driving Force in Solution Assays and Protein Film Voltammetry: Insights from the Comparison of Nitrate Reductase Mutants. Biochemistry 2010, 49, 2424−2432. (21) Fourmond, V.; Sabaty, M.; Arnoux, P.; Bertrand, P.; Pignol, D.; Leger, C. Reassessing the Strategies for Trapping Catalytic Intermediates during Nitrate Reductase Turnover. J. Phys. Chem. B 2010, 114, 3341−3347. (22) Coelho, C.; Marangon, J.; Rodrigues, D.; Moura, J. J. G.; Romão, M. J.; Paes de Sousa, P. M.; Correia dos Santos, M. M. Induced Peroxidase Activity of Haem Containing Nitrate Reductases

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CONCLUSIONS Electrochemically driven catalysis of the plant nitrate reductase from Arabidopsis thaliana has been studied for the first time using low redox potential mediators such as MV, BV, and AQ as the artificial enzyme redox partner. Both transient and sigmoidal forms of voltammograms were obtained depending upon mediator and substrate concentration, and a wide variety of waveforms were observed as a function of sweep rate and concentration. Digital simulation was used to explore the kinetics of the enzymatic reaction as a function of the substrate and mediator concentration at different sweep rates, and a single set of rate constants was obtained that modeled the experimental data.

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ASSOCIATED CONTENT

S Supporting Information *

Experimental and simulated cyclic voltammetry at different concentrations of nitrate and at different sweep rates. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge support from the Australian Research Council (DP120101465) and the German Science foundation (DFG, SFB635 TP05).

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REFERENCES

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Mediated Electrochemistry of Nitrate Reductase from Arabidopsis

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